Method and instrument for detecting biomolecular interactions

ABSTRACT

Method and apparatus for detecting biomolecular interactions. The use of labels is not required and the methods may be performed in a high-throughput manner. An instrument system for detecting a biochemical interaction on a biosensor. The system includes an array of detection locations comprises a light source for generating collimated white light. A beam splitter directs the collimated white light towards a surface of a sensor corresponding to the detector locations. A detection system includes an imaging spectrometer receiving the reflected light and generating an image of the reflected light.

A. PRIORITY

This application claims the benefit of U.S. provisional application No.60/244,312 filed Oct. 30, 2000; U.S. provisional application No.60/283,314 filed Apr. 12, 2001; U.S. provisional application No.60/303,028 filed Jul. 3, 2001; and is a continuation-in-part of U.S.patent application Ser. No. 09/930,352 filed Aug. 15, 2001, U.S. patentapplication Ser. No. 10/059,060 filed Jan. 28, 2002, and U.S. patentapplication Ser. No. 10/058,626 filed Jan. 28, 2002, all of which areherein entirely incorporated by reference and to which the reader isdirected for further information.

B. TECHNICAL AREA OF THE INVENTION

The invention generally relates to methods, instrumentation and devicesfor detecting biomolecular interactions.

C. BACKGROUND OF THE INVENTION

With the completion of the sequencing of the human genome, one of thenext grand challenges of molecular biology will be to understand how themany protein targets encoded by DNA interact with other proteins, smallmolecule pharmaceutical candidates, and a large host of enzymes andinhibitors. See e.g., Pandey & Mann, “Proteomics to study genes andgenomes,” Nature, 405, p. 837-846, 2000; Leigh Anderson et al.,“Proteomics: applications in basic and applied biology,” Current Opinionin Biotechnology, 11, p. 408-412, 2000; Patterson, “Proteomics: theindustrialization of protein chemistry,” Current Opinion inBiotechnology, 11, p. 413-418, 2000; MacBeath & Schreiber, “PrintingProteins as Microarrays for High-Throughput Function Determination,”Science, 289, p. 1760-1763, 2000; De Wildt et al., “Antibody arrays forhigh-throughput screening of antibody-antigen interactions,” NatureBiotechnology, 18, p. 989-994, 2000. To this end, tools that have theability to simultaneously quantify many different biomolecularinteractions with high sensitivity will find application inpharmaceutical discovery, proteomics, and diagnostics. Further, forthese tools to find widespread use, they must be simple to use,inexpensive to own and operate, and applicable to a wide range ofanalytes that can include, for example, polynucleotides, peptides, smallproteins, antibodies, and even entire cells.

Biosensors have been developed to detect a variety of biomolecularcomplexes including oligonucleotides, antibody-antigen interactions,hormone-receptor interactions, and enzyme-substrate interactions. Ingeneral, biosensors consist of two components: a highly specificrecognition element and a transducer that converts the molecularrecognition event into a quantifiable signal. Signal transduction hasbeen accomplished by many methods, including fluorescence,interferometry (Jenison et al., “Interference-based detection of nucleicacid targets on optically coated silicon,” Nature Biotechnology, 19, p.62-65; Lin et al., “A porous silicon-based optical interferometricbiosensor,” Science, 278, p. 840-843, 1997), and gravimetry (A.Cunningham, Bioanalytical Sensors, John Wiley & Sons (1998)).

Of the optically-based transduction methods, direct methods that do notrequire labeling of analytes with fluorescent compounds are of interestdue to the relative assay simplicity and ability to study theinteraction of small molecules and proteins that are not readilylabeled. Direct optical methods include surface plasmon resonance (SPR)(Jordan & Corn, “Surface Plasmon Resonance Imaging Measurements ofElectrostatic Biopolymer Adsorption onto Chemically Modified GoldSurfaces,” Anal. Chem., 69:1449-1456 (1997), (grating couplers (Morhardet al., “Immobilization of antibodies in micropatterns for celldetection by optical diffraction,” Sensors and Actuators B, 70, p.232-242, 2000), ellipsometry (Jin et al., “A biosensor concept based onimaging ellipsometry for visualization of biomolecular interactions,”Analytical Biochemistry, 232, p. 69-72, 1995), evanascent wave devices(Huber et al., “Direct optical immunosensing (sensitivity andselectivity),” Sensors and Actuators B, 6, p. 122-126, 1992), andreflectometry (Brecht & Gauglitz, “Optical probes and transducers,”Biosensors and Bioelectronics, 10, p. 923-936, 1995). Theoreticallypredicted detection limits of these detection methods have beendetermined and experimentally confirmed to be feasible down todiagnostically relevant concentration ranges. However, to date, thesemethods have yet to yield commercially available high-throughputinstruments that can perform high sensitivity assays without any type oflabel in a format that is readily compatible with the microtiterplate-based or microarray-based infrastructure that is most often usedfor high-throughput biomolecular interaction analysis. Therefore, thereis a need in the art for compositions and methods that can achieve thesegoals.

D. SUMMARY OF THE INVENTION

It is an object of the invention to provide methods, instrumentation anddevices for detecting binding of one or more specific binding substancesto their respective binding partners. This and other objects of theinvention are provided by one or more of the embodiments describedbelow.

In one arrangement, an instrument system for detecting a biochemicalinteraction on a biosensor comprising an array of detection locationscomprises a light source for generating collimated white light. A beamsplitter directs the collimated white light towards a surface of asensor corresponding to the detector locations. A detection systemincludes an imaging spectrometer receiving the reflected light andgenerating an image of the reflected light.

In an alternative arrangement, an instrument for calculating a peakwavelength comprises an incubator assembly for incubating a biosensor.An optical assembly illuminates the biosensor with light and collectsreflected radiation from the biosensor. A spectrometer receives the saidreflected radiation and software derives a peak wavelength from thereflected and detected wavelength.

Unlike surface plasmon resonance, resonant mirrors, and waveguidebiosensors, the described compositions and methods enable many thousandsof individual binding reactions to take place simultaneously upon thebiosensor surface. This technology is useful in applications where largenumbers of biomolecular interactions are measured in parallel,particularly when molecular labels alter or inhibit the functionality ofthe molecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by this approach. Abiosensor of the invention can be manufactured, for example, in largeareas using a plastic embossing process, and thus can be inexpensivelyincorporated into common disposable laboratory assay platforms such asmicrotiter plates and microarray slides.

These as well as other features and advantages of the present inventionwill become apparent to those of ordinary skill in the art by readingthe following detailed description, with appropriate reference to theaccompanying drawings.

E. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a schematic diagram of an embodiment of an opticalgrating structure.

FIG. 1B illustrates a perspective view of the optical grating structureillustrated in FIG. 1A.

FIG. 2 illustrates a schematic drawing of a linear grating structure.

FIG. 3A illustrates a 2-D biosensor grating comprising a grid ofsquares/rectangles.

FIG. 3B illustrates a 2-D biosensor grating comprising a grid ofcircular holes.

FIG. 4 illustrates an embodiment of a biosensor utilizing a sinusoidallyvarying grating profile.

FIG. 5(a) illustrates an alternative embodiment of a biosensor utilizingan embossed substrate.

FIG. 5(b) illustrates an alternative embodiment of a biosensor utilizinga plurality of concentric rings.

FIG. 5(c) illustrates an alternative embodiment of a biosensor having anarray of closely packed hexagons.

FIG. 6A illustrates a transparent resonant reflection superstructurearrangement.

FIG. 6B illustrates an alternative superstructure arrangement.

FIG. 6C illustrates a reflective surface of an alternative embodiment ofa biosensor.

FIG. 7 illustrates an alternative embodiment of a biosensor gratingstructure.

FIG. 8A illustrates a biosensor embodiment incorporated into amicrotiter plate.

FIG. 8B illustrates a microarray slide that may be utilized with themicrotiter plate embodiment illustrated in FIG. 8A.

FIG. 9 illustrates an embodiment of a biosensor platform to performassays.

FIG. 10 illustrates an embodiment of an array of biosensor electrodes.

FIG. 11 illustrates a SEM photograph showing a plurality of separategrating regions of an array of biosensor electrodes.

FIG. 12(a) illustrates an embodiment of a biosensor upper surfaceimmersed in a liquid sample.

FIG. 12(b) illustrates an attraction of electronegative molecules to abiosensor surface when a positive voltage is applied to the biosensorillustrated in FIG. 12(a).

FIG. 12(c) illustrates an application of a repelling force such as areversed electrical charge to electronegative molecules when a negativeelectrode voltage is applied to the biosensor illustrated in FIG. 12(a).

FIG. 13 illustrates an embodiment of a detecting system.

FIG. 14 illustrates resonance wavelength of a biosensor as a function ofincident angle of detection beam.

FIG. 15 illustrates an alternative embodiment of a detection system thatincludes a beam splitter.

FIG. 16 illustrates an embodiment of a biosensor incorporating an ITOgrating.

FIG. 17 illustrates an optical fiber probe measuring apparatus.

FIG. 18(a) illustrates an optical fiber probe that may be utilized withthe optical fiber probe measuring apparatus illustrated in FIG. 17.

FIG. 18(b) illustrates a general arrangement of a CCD chip and aspectrometer.

FIG. 18(c) illustrates a readout of a grating and the CCD chipillustrated in FIG. 18(b).

FIG. 19 illustrates an alternative embodiment of a measuring apparatus.

FIG. 20 illustrates yet another alternative embodiment of a measuringapparatus.

FIG. 21 illustrates an alternative embodiment of an instrument fordetection of biomolecular interactions in accordance with one possibleembodiment.

FIG. 22 illustrates a perspective view of the measuring apparatusillustrated in FIG. 21.

FIG. 23 illustrates a perspective view of the transition stage assemblyillustrated in FIG. 22.

FIG. 24 illustrates a perspective view of the transition stage assemblyof FIG. 23 with an incubator assembly top portion removed.

FIG. 25 illustrates a visualization of a spotted micro-array image.

F. DETAILED DESCRIPTION OF THE INVENTION

1. Overview of Method and System

The present invention generally relates to a method and system fordetecting biomolecular interactions. Preferably, these biomolecularinteractions occur on a subwavelength structured surface biosensor, asdescribed below.

One aspect of the present invention relates to a method and apparatusfor detecting biochemical interactions occurring on a surface of abiosensor. In one embodiment, the biosensor is a colorimetric resonantoptical biosensor embedded into a surface of a microarray slide,microtiter plate or other device.

The colorimetric resonant optical biosensor allows biochemicalinteractions to be measured on the sensor's surface without the use offluorescent tags or colorimetric labels. The sensor surface contains anoptical structure that, when illuminated with collimated white light, isdesigned to reflect only a narrow band of wavelengths. The narrowwavelength is described as a wavelength “peak.” The “peak wavelengthvalue” (PWV) changes when biological material is deposited or removedfrom the sensor surface.

A disclosed measurement instrument performs a number of functions, inaddition to detection of a peak wavelength value. For example, theinstrument can incubate a microtiter plate incorporating the biosensorplate at a user determined temperature. The instrument may also providea mechanism for mixing samples within the microtiter plate wells whilethe microtiter plate resides within the instrument.

In one possible embodiment, the instrument illuminates the biosensorsurface by directing a collimated white light on to the sensorstructure. The illuminated light may take the form of a spot ofcollimated light. Alternatively, the light is generated in the form of afan beam.

The instrument collects light reflected from the illuminated biosensorsurface. The instrument may gather this reflected light from multiplelocations on the biosensor surface simultaneously. The instrument caninclude a plurality of illumination probes that direct the light to adiscrete number of positions across the biosensor surface. Theinstrument measures the Peak Wavelength Values (PWVs) of separatelocations within the biosensor-embedded microtiter plate using aspectrometer. In one embodiment, the spectrometer is a single-pointspectrometer. Alternatively, an imaging spectrometer is used.

The spectrometer produces a PWV image map of the sensor surface. In oneembodiment, the measuring instrument spatially resolves PWV images withless than 200 micron resolution.

2. Subwavelength Structured Surface (SWS) Biosensor

In one embodiment of the present invention, a subwavelength structuredsurface (SWS) may be used to create a sharp optical resonant reflectionat a particular wavelength that can be used to track with highsensitivity the interaction of biological materials, such as specificbinding substances or binding partners or both. A colormetric resonantdiffractive grating surface acts as a surface binding platform forspecific binding substances.

Subwavelength structured surfaces are an unconventional type ofdiffractive optic that can mimic the effect of thin-film coatings. (Peng& Morris, “Resonant scattering from two-dimensional gratings,” J. Opt.Soc. Ain. A, Vol. 13, No. 5, p. 993, May; Magnusson, & Wang, “Newprinciple for optical filters,” Appl. Phys. Lett., 61, No. 9, p. 1022,August, 1992; Peng & Morris, “Experimental demonstration of resonantanomalies in diffraction from two-dimensional gratings,” Optics Letters,Vol. 21, No. 8, p. 549, April, 1996). A SWS structure contains asurface-relief, two-dimensional grating in which the grating period issmall compared to the wavelength of incident light so that nodiffractive orders other than the reflected and transmitted zerothorders are allowed to propagate. A SWS surface narrowband filter cancomprise a two-dimensional grating sandwiched between a substrate layerand a cover layer that fills the grating grooves. Optionally, a coverlayer is not used. When the effective index of refraction of the gratingregion is greater than the substrate or the cover layer, a waveguide iscreated.

When a filter is designed according to one aspect of the presentinvention, incident light passes into the waveguide region. Atwo-dimensional grating structure selectively couples light at a narrowband of wavelengths into the waveguide. The light propagates only ashort distance (on the order of 10-100 micrometers), undergoesscattering, and couples with the forward- and backward-propagatingzeroth-order light. This sensitive coupling condition can produce aresonant grating effect on the reflected radiation spectrum, resultingin a narrow band of reflected or transmitted wavelengths. The depth andperiod of the two-dimensional grating are less than the wavelength ofthe resonant grating effect.

The reflected or transmitted color of this structure can be modulated bythe addition of molecules such as specific binding substances or bindingpartners or both to the upper surface of the cover layer or thetwo-dimensional grating surface. The added molecules increase theoptical path length of incident radiation through the structure, andthus modify the wavelength at which maximum reflectance or transmittancewill occur.

In one embodiment, a biosensor, when illuminated with white light, isdesigned to reflect only a single wavelength. When specific bindingsubstances are attached to the surface of the biosensor, the reflectedwavelength (color) is shifted due to the change of the optical path oflight that is coupled into the grating. By linking specific bindingsubstances to a biosensor surface, complementary binding partnermolecules can be detected without the use of any kind of fluorescentprobe or particle label. The detection technique is capable of resolvingchanges of, for example, ˜0.1 nm thickness of protein binding, and canbe performed with the biosensor surface either immersed in fluid ordried. A more detailed description of these binding partners is providedin related and commonly assigned patent application Ser. No. 09/930,352,herein entirely incorporated by reference and to which the reader isdirected for further information.

In one embodiment of the present invention, a detection system consistsof, for example, a light source that illuminates a small spot of abiosensor at normal incidence through, for example, a fiber optic probe.A spectrometer collects the reflected light through, for example, asecond fiber optic probe also at normal incidence. Because no physicalcontact occurs between the excitation/detection system and the biosensorsurface, no special coupling prisms are required. The biosensor can,therefore, be adapted to a commonly used assay platform including, forexample, microtiter plates and microarray slides. A spectrometer readingcan be performed in several milliseconds, thus it is possible toefficiently measure a large number of molecular interactions takingplace in parallel upon a biosensor surface, and to monitor reactionkinetics in real time.

This technology is useful in applications where large numbers ofbiomolecular interactions are measured in parallel, particularly whenmolecular labels would alter or inhibit the functionality of themolecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by the compositionsand methods of the invention.

A schematic diagram of an example of a SWS structure 10 is shown inFIG. 1. In FIG. 1, n_(substrate) represents a refractive index of asubstrate material 12. n₁ represents the refractive index of an optionalcover layer 14. n₂ represents the refractive index of a two-dimensionalgrating 16. N_(bio) represents the refractive index of one or morespecific binding substances 20. t₁ represents the thickness of the coverlayer 14 above the two-dimensional grating structure 16. t₂ representsthe thickness of the grating. t_(bio) represents the thickness of thelayer of one or more specific binding substances 20. In one embodiment,n2>n1 (see FIG. 1). Layer thicknesses (i.e. cover layer 14, one or morespecific binding substances 20, or a two-dimensional grating 16) areselected to achieve resonant wavelength sensitivity to additionalmolecules on a top surface 15. A grating period is selected to achieveresonance at a desired wavelength.

One embodiment provides a SWS biosensor, such as the SWS biosensor 10illustrated in FIG. 1. The SWS biosensor 10 comprises a two-dimensionalgrating 16 and a substrate layer 12 that supports the two-dimensionalgrating 16. One or more specific binding substances 20 may beimmobilized on a surface 15 of the two-dimensional grating 16 oppositeof the substrate layer 12. Incident light 11 is polarized perpendicularto the grating structure and results in the reflected light 13. FIG. 1Billustrates a perspective view of the optical grating structureillustrated in FIG. 1A. Note that a SWS biosensor works equally wellwhether illumination and reflection occur from the top of the sensorsurface or from the bottom, although FIG. 1 illustrates only the bottomillumination and reflection case.

The two-dimensional grating 16 can comprise a material, including, forexample, zinc sulfide, titanium dioxide, tantalum oxide, and siliconnitride. A cross-sectional profile of a two-dimensional grating cancomprise any periodically repeating function, for example, a“square-wave.” The two-dimensional grating can comprise a repeatingpattern of shapes selected from the group consisting of lines, squares,circles, ellipses, triangles, trapezoids, sinusoidal waves, ovals,rectangles, and hexagons. A sinusoidal cross-sectional profile ispreferable for manufacturing applications that require embossing of agrating shape into a soft material such as plastic. In one embodiment ofthe biosensor, the depth of the grating is about 0.01 micron to about 1micron and the period of the grating is about 0.01 micron to about 1micron.

Linear gratings have resonant characteristics where the illuminatinglight polarization is oriented perpendicular to the grating period.However, a hexagonal grid of holes has increased polarization symmetryover that of a rectangular grid of holes. Therefore, a colorimetricresonant reflection biosensor can comprise, for example, a hexagonalarray of holes or alternatively, a grid of parallel lines. FIG. 3Billustrates a 2-D biosensor grating 34 including a grid of circularholes 36. FIG. 3A illustrates a 2-D biosensor grating 30 including agrid of squares/rectangles 32.

A linear grating has the same pitch (i.e. distance between regions ofhigh and low refractive index), period, layer thicknesses, and materialproperties as the hexagonal array grating. However, light must bepolarized perpendicular to the grating lines in order to be resonantlycoupled into the optical structure. Therefore, a polarizing filteroriented with its polarization axis perpendicular to the linear gratingis inserted between the illumination source and the biosensor surface.Because only a small portion of the illuminating light source iscorrectly polarized, a longer integration time is required to collect anequivalent amount of resonantly reflected light compared to a hexagonalgrating.

While a linear grating can require either a higher intensityillumination source or a longer measurement integration time compared toa hexagonal grating, the fabrication requirements for the linearstructure are generally less complex. A hexagonal grating pattern isproduced by holographic exposure of photoresist to three mutuallyinterfering laser beams. The three beams are aligned in order to producea grating pattern that is essentially symmetrical in three directions. Alinear grating pattern requires alignment of only two laser beams toproduce a holographic exposure in photoresist, and therefore has areduced alignment requirement. A linear grating pattern can also beproduced by, for example, direct writing of photoresist with an electronbeam. Also, several commercially available sources exist for producinglinear grating “master” templates for embossing a grating structure intoplastic.

A schematic diagram of a linear grating structure 24 is shown in FIG. 2.The grating structure 24 includes a grating fill layer 25, a gratingstructural layer 26, and a substrate 27. Incident light 28 is polarizedperpendicular to the grating structure and results in the reflectedlight 29.

A rectangular grid pattern can be produced in photoresist using anelectron beam direct-write exposure system. A single wafer can beilluminated as a linear grating with two sequential exposures with thepart rotated 90-degrees between exposures.

FIG. 5(a) illustrates a two-dimensional grating, 50. Two-dimensionalgrating 50 comprises a “stepped” profile 52. In such a stepped profile52, the profile has high refractive index regions of a single, fixedheight embedded within a lower refractive index cover layer. Thealternating regions of high and low refractive index provide an opticalwaveguide parallel to a top surface of the biosensor.

For manufacture, a stepped structure, such as the structure illustratedin FIG. 5(a), is etched or embossed into a substrate material 54 such asglass or plastic. A uniform thin film of higher refractive indexmaterial 51, such as silicon nitride (SiN) or zinc sulfide (ZnS) isdeposited on this structure. The deposited layer follows the contour ofthe embossed or etched structure in the substrate 54. Consequently, thedeposited material 51 has a surface relief profile that is essentiallyidentical to the original embossed or etched profile of embossed plasticsubstrate 54.

The structure 50 can be completed with an application of an optionalcover layer 56. Preferably, cover layer 56 includes a material having alower refractive index than the higher refractive index material and hasa substantially flat upper surface 57. The covering material 56 can be,for example, glass, epoxy, or plastic.

A stepped structure, such as the structure illustrated in FIG. 5(a),allows for reduced cost biosensor manufacturing, because the biosensorcan be mass produced. For example, a “master” grating can be produced inglass, plastic, or metal using a three-beam laser holographic patterningprocess. See e.g., Cowan, The recording and large scale production ofcrossed holographic grating arrays using multiple beam interferometry,Proc. Soc. Photo-optical Instum. Eng. 503:120 (1984). A master gratingcan be repeatedly used to emboss a plastic substrate. The embossedsubstrate 50 is subsequently coated with a high refractive indexmaterial and optionally, a cover layer 56.

While a stepped structure poses essentially few manufacturingcomplications, it is also possible to make a resonant biosensor in whicha high refractive index material is not stepped. Rather, a biosensorcould include a high refractive index material that varies with lateralposition. For example, FIG. 4 illustrates an alternative embodiment of abiosensor 40. In this embodiment, the biosensor 40 includes atwo-dimensional grating 42 having a high refractive index materialvarying with lateral position.

The biosensor 40 includes a substrate layer 41 that supports thetwo-dimensional grating 42. One or more specific binding substances 46may be immobilized on a surface 43 of the two-dimensional gratingopposite of the substrate layer 41. Sensor 40 includes a profile inwhich a high refractive index material of the two-dimensional grating42, n₂, sinusoidally varies in height. This varying height of thetwo-dimensional grating 42 is represented by t₂.

To produce a resonant reflection at a particular wavelength, the periodof the sinusoidally varying grating 42 is essentially identical to theperiod of an equivalent stepped structure. The resonant operation of thesinusoidally varying structure 40 and its functionality as a biosensorhas been verified using GSOLVER (Grating Solver Development Company,Allen, Tex., USA) computer models.

Techniques for making two-dimensional gratings are disclosed in Wang, J.Opt. Soc. Am No. 8, August 1990, pp. 1529-44. Biosensors as hereindescribed can be made in, for example, a semiconductor microfabricationfacility. Biosensors can also be made on a plastic substrate usingcontinuous embossing and optical coating processes. For this type ofmanufacturing process, a “master” structure is built in a rigid materialsuch as glass or silicon. These “master” structures are used to generate“mother” structures in an epoxy or plastic using one of several types ofreplication procedures. The “mother” structure, in turn, is coated witha thin film of conductive material, and used as a mold to electroplate athick film of nickel. The nickel “daughter” is released from the plastic“mother” structure. Finally, the nickel “daughter” is bonded to acylindrical drum, which is used to continuously emboss the surfacerelief structure into a plastic film.

A device structure that uses an embossed plastic substrate is shown inFIG. 5(a). Following embossing, the plastic substrate 54 is overcoatedwith a thin film of high refractive index material 51. The substrate 54may be optionally coated with a planarizing, cover layer polymer, andcut to appropriate size.

In one biosensor embodiment, a substrate for a SWS biosensor comprisesglass, plastic or epoxy. Alternatively, a substrate and atwo-dimensional grating comprise a single biosensor unit. That is, a twodimensional grating and substrate are formed from the same material,such as, for example, glass, plastic, or epoxy. The surface of a singleunit comprising the two-dimensional grating is coated with a materialhaving a high refractive index, for example, zinc sulfide, titaniumdioxide, tantalum oxide, and silicon nitride. One or more specificbinding substances can be immobilized on the surface of the materialhaving a high refractive index or on an optional cover layer.

An alternative biosensor embodiment can further comprise a cover layeron the surface of a two-dimensional grating opposite of a substratelayer. Where a cover layer is present, the one or more specific bindingsubstances are immobilized on the surface of the cover layer opposite ofthe two-dimensional grating. Preferably, a cover layer comprises amaterial that has a lower refractive index than a material thatcomprises the two-dimensional grating. A cover layer can be comprisedof, for example, glass (including spin-on glass (SOG)), epoxy, orplastic.

Various polymers that meet the refractive index requirement of abiosensor can be used for a cover layer. For example, SOG can be useddue to its favorable refractive index, ease of handling, and readinessof being activated with specific binding substances using a number ofknown glass surface activation techniques. When the flatness of thebiosensor surface is not an issue for a particular application, agrating structure of SiN/glass can directly be used as the sensingsurface, the activation of which can be done using the same means as ona glass surface.

Resonant reflection can also be obtained without a planarizing coverlayer over a two-dimensional grating. For example, a biosensor cancontain only a substrate coated with a structured thin film layer ofhigh refractive index material. Without the use of a planarizing coverlayer, the surrounding medium (such as air or water) fills the grating.Therefore, specific binding substances are immobilized to the biosensoron all surfaces a two-dimensional grating exposed to the specificbinding substances, rather than only on an upper surface.

According to an alternative embodiment, a biosensor is illuminated withwhite light that contains light of every polarization angle. Theorientation of the polarization angle with respect to repeating featuresin a biosensor grating determines a resonance wavelength. For example, a“linear grating” biosensor structure consisting of a set of repeatinglines and spaces will have two optical polarizations that can generateseparate resonant reflections. Light that is polarized perpendicularlyto the lines is referred to as “s-polarized,” whereas light that ispolarized parallel to the lines is referred to as “p-polarized.” Boththe s and the p components of incident light exist simultaneously in anunfiltered illumination beam, and each component generates a separateresonant signal. A biosensor structure can generally be designed tooptimize the properties of only one polarization (the s-polarization),and the non-optimized polarization can be removed by a polarizingfilter.

In order to remove the polarization dependence, so that everypolarization angle generates the same resonant reflection spectra, analternate biosensor structure can be used. Such an alternative biosensorcould consist of a plurality of concentric rings, such as the structureillustrated in FIG. 5(b). In this structure, the difference between aninside diameter and an outside diameter of each concentric ring is equalto about one-half of a grating period. Each successive ring has aninside diameter that is about one grating period greater than the insidediameter of the previous ring. The concentric ring pattern extends tocover a single sensor location—such as a microarray spot or a microtiterplate well. Each separate microarray spot or microtiter plate well has aseparate concentric ring pattern centered within it.

All polarization directions of such a structure have the samecross-sectional profile. The concentric ring structure, such as thestructure illustrated in FIG. 5(b), should be illuminated on-center topreserve polarization independence. The grating period of a concentricring structure is preferably less than the wavelength of the resonantlyreflected light. In one preferred embodiment, the grating period isabout 0.01 micron to about 1 micron and the grating depth is about 0.01to about 1 micron.

In another biosensor embodiment, an array of holes or posts are providedon a sensor surface, such as the design illustrated in FIG. 5(c).Preferably, the array of holes or posts are arranged to approximate theconcentric circle structure described above without requiring theillumination beam to be centered upon any particular location of thegrid. Such an array pattern is automatically generated by the opticalinterference of three laser beams incident on a surface from threedirections at equal angles. In this pattern, the holes (or posts) arecentered upon the corners of an array of closely packed hexagons, asillustrated in FIG. 5(c). The holes or posts also occur in the center ofeach hexagon. Such a hexagonal grid of holes or posts has threepolarization directions that “see” the same cross-sectional profile. Thehexagonal grid structure, therefore, provides equivalent resonantreflection spectra using light of any polarization angle. Thus, nopolarizing filter is required to remove unwanted reflected signalcomponents. The period of the holes or posts can be about 0.01 micronsto about 1 micron and the depth or height can be about 0.01 microns toabout 1 micron. These and various other grid structures are disclosedand described in commonly assigned co-pending patent application Ser.No. 09/930,352 herein entirely incorporated by reference and to whichthe reader is directed for further details.

One possible detection apparatus and method provides for resonantreflection structures and transmission filter structures comprisingconcentric circle gratings and hexagonal grids of holes or posts. For aresonant reflection structure, light output is measured on the same sideof the structure as the illuminating light beam. For example, asillustrated in FIG. 2, reflected light 29 is measured on the same sideof the structure 21 as the illuminating light beam or incident light 28.

For a transmission filter structure, light output is measured on theopposite side of the structure as the illuminating beam. The reflectedand transmitted signals are complementary. That is, if a wavelength isstrongly reflected, it is weakly transmitted. Assuming no energy isabsorbed in the structure itself, the combined reflected energy andtransmitted energy at a given wavelength will remain a constant. Theresonant reflection structure and transmission filters are designed toprovide an efficient reflection at a specified wavelength. Therefore, areflection filter will “pass” a narrow band of wavelengths, while atransmission filter will filter out or “cut” a narrow band ofwavelengths from incident light.

A resonant reflection structure or a transmission filter structure cancomprise a two-dimensional grating arranged in a pattern of concentriccircles. (See, e.g., FIG. 5(b). A resonant reflection structure ortransmission filter structure can also comprise a hexagonal grid ofholes or posts. (See, e.g., FIG. 5(c)).

When these structures are illuminated with an illuminating light beam, areflected radiation spectrum is produced. Such a radiation spectrum isindependent of an illumination polarization angle of the illuminatinglight beam. A resonant grating effect is produced on the reflectedradiation spectrum, wherein the depth and period of the two-dimensionalgrating or hexagonal grid of holes or posts are less than the wavelengthof the resonant grating effect. These structures reflect a narrow bandof light when the structure is illuminated with a broadband of light.

Resonant reflection structures and transmission filter structures of theinvention can be used as biosensors. For example, one or more specificbinding substances can be immobilized on the hexagonal grid of holes orposts or on the two-dimensional grating arranged in concentric circles.

In an alternative embodiment, a reference resonant signal is providedfor more accurate measurement of peak resonant wavelength shifts. Thereference resonant signal can cancel out environmental effects,including, for example, temperature or other types of unwanted noise. Areference signal can be provided using a resonant reflectionsuperstructure that produces two separate resonant wavelengths. Forexample, FIG. 6A illustrates a transparent resonant reflectionsuperstructure arrangement 60. Transparent resonant reflectionsuperstructure 60 contains two sub-structures. The sub-structurescomprise a low n cured polymer 66 and a high n dielectric 67. A firstsub-structure comprises a first two-dimensional grating 62 with a top 61a and a bottom surface 61 b. The top surface of a two-dimensionalgrating 61 a comprises the grating surface 63. The first two-dimensionalgrating can comprise one or more specific binding substances immobilizedon its top surface. The top surface of the first two-dimensional grating63 will be in contact with a test sample (not shown). An optionalsubstrate layer 64 may provide support to the bottom surface 61 b of thefirst two-dimensional grating. The substrate layer 64 comprises a top 65a and bottom surface 65 b. The top surface 65 a of the substrate 64 isin contact with, and supports the bottom surface 61 b of the firsttwo-dimensional grating 62. A low n substrate 68 is also provided.

A second sub-structure 70, illustrated in FIG. 6B, comprises a secondtwo-dimensional grating 72 with a top surface 73 a and a bottom surface73 b. The second two-dimensional grating is not in contact with a testsample. The sub-structures comprise a low n cured polymer 77 and a highn dielectric 71. The second two-dimensional grating can be fabricatedonto a bottom surface of the substrate 74 that supports a firsttwo-dimensional grating 76. Where the second two-dimensional grating isfabricated on the substrate that supports the first two-dimensionalgrating, the bottom surface 73 a of the second two-dimensional grating72 can be fabricated onto the bottom surface 75 of the substrate 74.Therefore, the top surface 73 b of the second two-dimensional grating 72will face the opposite direction of a surface 77 a of the firsttwo-dimensional grating 76.

The top surface 73 b of the second two-dimensional grating 72 can alsobe attached directly to the bottom surface of the first sub-structure.In this arrangement, the top surface of the second two-dimensionalgrating would face in the same direction as the top surface 77 a of thefirst two-dimensional grating 76. A substrate 74 can support the bottomsurface of the second two-dimensional grating in this arrangement.

Because the second sub-structure is not in physical contact with thetest sample, its peak resonant wavelength is not subject to changes inthe optical density of the test media, or deposition of specific bindingsubstances or binding partners on the surface of the firsttwo-dimensional grating. Therefore, such a superstructure produces tworesonant signals. Because the location of the peak resonant wavelengthin the second sub-structure is fixed, the difference in peak resonantwavelength between the two sub-structures provides a relative means fordetermining the amount of specific binding substances or bindingpartners or both deposited on the top surface of the first substructurethat is exposed to the test sample.

A biosensor superstructure can be illuminated from its top surface orfrom its bottom surface, or from both surfaces. The peak resonancereflection wavelength of the first substructure is dependent on theoptical density of material in contact with the superstructure surface.The peak resonance reflection wavelength of the second substructure isindependent of the optical density of material in contact with thesuperstructure surface.

In an alternative embodiment, a biosensor is illuminated from a bottomsurface. Approximately 50% of the incident light is reflected from thebottom surface of biosensor without reaching the active (or the top)surface of the biosensor. A thin film or physical structure can beincluded in a biosensor composition that is capable of maximizing theamount of light that is transmitted to the upper surface of thebiosensor while minimizing the reflected energy at the resonantwavelength. The anti-reflection thin film or physical structure of thebottom surface of the biosensor can comprise, for example, a singledielectric thin film, or a stack of multiple dielectric thin films.

Alternatively, a “motheye” structure embossed into the bottom biosensorsurface is provided. An example of a motheye structure is disclosed inHobbs, et al. “Automated interference lithography system for generationof sub-micron feature size patterns,” Proc. 1999 Micromachine Technologyfor Diffracting and Holographic Optics, Society of Photo-OpticalInstrumentation Engineers, p. 124135, (1999).

In one embodiment of the present invention, an interaction of a firstmolecule with a second test molecule is detected. A SWS biosensor aspreviously described is used. However, there are no specific bindingsubstances immobilized on a SWS biosensor. Therefore, the biosensorcomprises a two-dimensional grating, a substrate layer that supports thetwo-dimensional grating. Optionally, a cover layer may be provided. Asdescribed above, when the biosensor is illuminated a resonant gratingeffect is produced on the reflected radiation spectrum, and the depthand period of the two-dimensional grating are less than the wavelengthof the resonant grating effect.

To detect an interaction of a first molecule with a second testmolecule, a mixture of the first and second molecules is applied to adistinct location on a biosensor. Such a location may be one area, spot,or one well on the biosensor. Alternatively, it could be a large area onthe biosensor. A mixture of the first molecule with a third controlmolecule is also applied to a distinct location on a biosensor. Thebiosensor can be the same biosensor as described above, or can be asecond biosensor. If the biosensor is the same biosensor, a seconddistinct location can be used for the mixture of the first molecule andthe third control molecule.

Alternatively, the same distinct biosensor location can be used afterthe first and second molecules are washed from the biosensor. The thirdcontrol molecule does not interact with the first molecule and may beabout the same size as the first molecule. A shift in the reflectedwavelength of light from the distinct locations of the biosensor orbiosensors is measured using a read out method and apparatus as detailedbelow.

If the shift in the reflected wavelength of light from the distinctlocation having the first molecule and the second test molecule isgreater than the shift in the reflected wavelength from the distinctlocation having the first molecule and the third control molecule, thenthe first molecule and the second test molecule interact. Interactioncan be, for example, hybridization of nucleic acid molecules, specificbinding of an antibody or antibody fragment to an antigen, and bindingof polypeptides. A first molecule, second test molecule, or thirdcontrol molecule can be, for example, a nucleic acid, polypeptide,antigen, polyclonal antibody, monoclonal antibody, single chain antibody(scFv), F(ab) fragment, F(ab′)₂ fragment, Fv fragment, small organicmolecule, cell, virus, and bacteria.

3. Specific Binding Substances and Binding Partners

One or more specific binding substances may be immobilized on thetwo-dimensional grating or cover layer, if present. Immobilization mayoccur by physical adsorption or by chemical binding. A specific bindingsubstance can be, for example, a nucleic acid, polypeptide, antigen,polyclonal antibody, monoclonal antibody, single chain antibody (scFv),F(ab) fragment, F(ab′)₂ fragment, Fv fragment, small organic molecule,cell, virus, bacteria, or biological sample. A biological sample can befor example, blood, plasma, serum, gastrointestinal secretions,homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum,cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lunglavage fluid, semen, lymphatic fluid, tears, or prostatitc fluid.

Preferably, one or more specific binding substances are arranged in amicroarray of distinct locations on a biosensor. A microarray ofspecific binding substances comprises one or more specific bindingsubstances on a surface of a biosensor such that a biosensor surfacecontains a plurality of distinct locations, each with a differentspecific binding substance or with a different amount of a specificbinding substance. For example, an array can comprise 1, 10, 100, 1,000,10,000, or 100,000 distinct locations. A biosensor surface with a largenumber of distinct locations is called a microarray because one or morespecific binding substances are typically laid out in a regular gridpattern in x-y coordinates. However, a microarray can comprise one ormore specific binding substances laid out in a regular or irregularpattern. For example, distinct locations can define a microarray ofspots of one or more specific binding substances.

A microarray spot can range from about 50 to about 500 microns indiameter. Alternatively, a microarray spot can range from about 150 toabout 200 microns in diameter. One or more specific binding substancescan be bound to their specific binding partners.

In one biosensor embodiment, a microarray on a biosensor is created byplacing microdroplets of one or more specific binding substances onto,for example, an x-y grid of locations on a two-dimensional grating orcover layer surface. When the biosensor is exposed to a test samplecomprising one or more binding partners, the binding partners will bepreferentially attracted to distinct locations on the microarray thatcomprise specific binding substances that have high affinity for thebinding partners. Some of the distinct locations will gather bindingpartners onto their surface, while other locations will not.

A specific binding substance specifically binds to a binding partnerthat is added to the surface of a biosensor of the invention. A specificbinding substance specifically binds to its binding partner, but doesnot substantially bind other binding partners added to the surface of abiosensor. For example, where the specific binding substance is anantibody and its binding partner is a particular antigen, the antibodyspecifically binds to the particular antigen, but does not substantiallybind other antigens. A binding partner can be, for example, a nucleicacid, polypeptide, antigen, polyclonal antibody, monoclonal antibody,single chain antibody (scFv), F(ab) fragment, F(ab′)₂ fragment, Fvfragment, small organic molecule, cell, virus, bacteria, and biologicalsample. A biological sample can be, for example, blood, plasma, serum,gastrointestinal secretions, homogenates of tissues or tumors, synovialfluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinalfluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid,tears, and prostatitc fluid.

In an alternative embodiment, a nucleic acid microarray is provided, inwhich each distinct location within the array contains a differentnucleic acid molecule. In this embodiment, the spots within the nucleicacid microarray detect complementary chemical binding with an opposingstrand of a nucleic acid in a test sample.

While microtiter plates are a common format used for biochemical assays,microarrays are increasingly seen as a means for increasing the numberof biochemical interactions that can be measured at one time whileminimizing the volume of precious reagents. By application of specificbinding substances with a microarray spotter onto a biosensor of theinvention, specific binding substance densities of 10,000 specificbinding substances/in² can be obtained. By focusing an illumination beamof a fiber optic probe to interrogate a single microarray location, abiosensor can be used as a label-free microarray readout system.

4. Immobilization of One or More Specific Binding Substances

Immobilization of one or more binding substances onto a biosensor isperformed so that a specific binding substance will not be washed awayby rinsing procedures, and so that its binding to binding partners in atest sample is unimpeded by the biosensor surface. Several differenttypes of surface chemistry strategies have been implemented for covalentattachment of specific binding substances to, for example, glass for usein various types of microarrays and biosensors. These methods can beadapted to a biosensor embodiment. Surface preparation of a biosensor sothat it contains certain required functional groups for binding one ormore specific binding substances can be an integral part of thebiosensor manufacturing process.

One or more specific binding substances can be attached to a biosensorsurface by physical adsorption (i.e., without the use of chemicallinkers). Alternatively, one or more specific binding substances can beattached to a biosensor surface by chemical binding (i.e., with the useof chemical linkers). Chemical binding can generate stronger attachmentof specific binding substances on a biosensor surface. Chemical bindingalso provides defined orientation and conformation of the surface-boundmolecules. Several examples of chemical binding of specific bindingsubstances to a biosensor embodying one aspect of the invention aredescribed in detail in related commonly assigned co-pending patentapplication Ser. No. 09/930,352 herein entirely incorporated byreference and to which the reader is directed for further detail.

Other types of chemical binding include, for example, amine activation,aldehyde activation, and nickel activation. These surfaces can be usedto attach several different types of chemical linkers to a biosensorsurface. While an amine surface can be used to attach several types oflinker molecules, an aldehyde surface can be used to bind proteinsdirectly, without an additional linker. A nickel surface can be used tobind molecules that have an incorporated histidine (“his”) tag.Detection of “his-tagged” molecules with a nickel-activated surface iswell known in the art (Whitesides, Anal. Chem. 68, 490, (1996)).

Immobilization of specific binding substances to plastic, epoxy, or highrefractive index material can be performed essentially as described forimmobilization to glass. However, an acid wash step may be eliminatedwhere such a treatment would damage the material to which the specificbinding substances are immobilized.

For the detection of binding partners at concentrations of less thanabout ˜0.1 ng/ml, one may amplify and transduce binding partners boundto a biosensor into an additional layer on the biosensor surface. Theincreased mass deposited on the biosensor can be detected as aconsequence of increased optical path length. By incorporating greatermass onto a biosensor surface, an optical density of binding partners onthe surface is also increased, thus rendering a greater resonantwavelength shift than would occur without the added mass. The additionof mass can be accomplished, for example, enzymatically, through a“sandwich” assay, or by direct application of mass to the biosensorsurface in the form of appropriately conjugated beads or polymers ofvarious size and composition. This principle has been exploited forother types of optical biosensors to demonstrate sensitivity increasesover 1500× beyond sensitivity limits achieved without massamplification. See, e.g., Jenison et al., “Interference-based detectionof nucleic acid targets on optically coated silicon,” NatureBiotechnology, 19: 62-65, 2001.

5. Surface-Relief Volume Diffractive Biosensors

In an alternative embodiment, a biosensor comprises volumesurface-relief volume diffractive structures (a SRVD biosensor). SRVDbiosensors have a surface that reflects predominantly at a particularnarrow band of optical wavelengths when illuminated with a broad band ofoptical wavelengths. Where specific binding substances and/or bindingpartners are immobilized on a SRVD biosensor, the reflected wavelengthof light is shifted. One-dimensional surfaces, such as thin filminterference filters and Bragg reflectors, can select a narrow range ofreflected or transmitted wavelengths from a broadband excitation source.However, the deposition of additional material, such as specific bindingsubstances and/or binding partners onto their upper surface results onlyin a change in the resonance linewidth, rather than the resonancewavelength. In contrast, SRVD biosensors have the ability to alter thereflected wavelength with the addition of material, such as specificbinding substances and/or binding partners to the surface.

A SRVD biosensor comprises a sheet material having a first and secondsurface. The first surface of the sheet material defines relief volumediffraction structures. Sheet material can comprise, for example,plastic, glass, semiconductor wafer, or metal film.

A relief volume diffractive structure can be, for example, atwo-dimensional grating, as described above, or a three-dimensionalsurface-relief volume diffractive grating. The depth and period ofrelief volume diffraction structures are less than the resonancewavelength of light reflected from a biosensor.

A three-dimensional surface-relief volume diffractive grating can be,for example, a three-dimensional phase-quantized terraced surface reliefpattern whose groove pattern resembles a stepped pyramid. When such agrating is illuminated by a beam of broadband radiation, light will becoherently reflected from the equally spaced terraces at a wavelengthgiven by twice the step spacing times the index of refraction of thesurrounding medium. Light of a given wavelength is resonantly diffractedor reflected from the steps that are a half-wavelength apart, and with abandwidth that is inversely proportional to the number of steps. Thereflected or diffracted color can be controlled by the deposition of adielectric layer so that a new wavelength is selected, depending on theindex of refraction of the coating.

A stepped-phase structure can be produced first in photoresist bycoherently exposing a thin photoresist film to three laser beams, asdescribed previously. See e.g., Cowen, “The recording and large scalereplication of crossed holographic grating arrays using multiple beaminterferometry,” in International Conference on the Application, Theory,and Fabrication of Periodic Structures, Diffraction Gratings, and MoirePhenomena II, Lerner, ed., Proc. Soc. Photo-Opt. Instrum. Eng., 503,120-129, 1984; Cowen, “Holographic honeycomb microlens,” Opt. Eng. 24,796-802 (1985); Cowen & Slafer, “The recording and replication ofholographic micropatterns for the ordering of photographic emulsiongrains in film systems,” J. Imaging Sci. 31, 100-107, 1987. Thenonlinear etching characteristics of photoresist are used to develop theexposed film to create a three-dimensional relief pattern. Thephotoresist structure is then replicated using standard embossingprocedures. For example, a thin silver film may be deposited over thephotoresist structure to form a conducting layer upon which a thick filmof nickel can be electroplated. The nickel “master” plate is then usedto emboss directly into a plastic film, such as vinyl, that has beensoftened by heating or solvent.

A theory describing the design and fabrication of three-dimensionalphase-quantized terraced surface relief pattern that resemble steppedpyramids is described: Cowen, “Aztec surface-relief volume diffractivestructure,” J. Opt. Soc. Am. A, 7:1529 (1990).

An example of a three-dimensional phase-quantized terraced surfacerelief pattern may be a pattern that resembles a stepped pyramid. Eachinverted pyramid is approximately 1 micron in diameter. Preferably, eachinverted pyramid can be about 0.5 to about 5 microns diameter, includingfor example, about 1 micron. The pyramid structures can be close-packedso that a typical microarray spot with a diameter of 150-200 microns canincorporate several hundred stepped pyramid structures. The reliefvolume diffraction structures have a period of about 0.1 to about 1micron and a depth of about 0.1 to about 1 micron.

FIG. 6 illustrates a reflective surface 82 of a biosensor 80. Thereflective surface 82 includes a first microarray spot 84 and a secondmicroarray spot 86. The first microarray spot 82 is not provided with anadsorbed material while the second microarray spot 84 is provided withan adsorbed protein. Consequently, the index or refraction for the firstspot remains unchanged, i.e., n_(air)=1. The index of refraction for thesecond spot will change to that of an absorbed protein, i.e.,n_(protein)=1.4.

As white light 81 is illuminated onto the first microarray spot 84, bluelight 89 will be reflected. Alternatively, the reflected light of whitelight 88 illuminated unto the second microarray spot 86 will bedifferent (i.e., green light 87) because of the different index ofrefraction.

FIG. 6 demonstrates how individual microarray locations (with an entiremicroarray spot incorporating hundreds of pyramids now represented by asingle pyramid for one microarray spot) can be optically queried todetermine if specific binding substances or binding partners areadsorbed onto the surface. When the structure is illuminated with whitelight, structures without significant bound material will reflectwavelengths determined by the step height of the structure. When higherrefractive index material, such as binding partners or specific bindingsubstances, are incorporated over the reflective metal surface, thereflected wavelength is modified to shift toward longer wavelengths. Thecolor that is reflected from the terraced step structure istheoretically given as twice the step height times the index ofrefraction of a reflective material that is coated onto the firstsurface of a sheet material of a SRVD biosensor. A reflective materialcan be, for example silver, aluminum, or gold.

One or more specific binding substances, as described above, areimmobilized on the reflective material of a SRVD biosensor. One or morespecific binding substances can be arranged in microarray of distinctlocations, as described above, on the reflective material. FIG. 7illustrates an embodiment of a microarray sensor 90. In this embodiment,the microarray biosensor 90 comprises a 9-element microarray biosensor.A plurality of individual grating structures, represented in FIG. 7 bysmall circles, such as small circle 91, lie within each microarray spot.The microarray spots, represented by the larger circles 92(a-i), reflectwhite light at a specific wavelength. This specific wavelength isdetermined by the refractive index of material on the microarraysurface. Microarray locations with additional adsorbed material willhave reflected wavelengths that are shifted toward longer wavelengths,represented by the larger circles.

Because the reflected wavelength of light from a SRVD biosensor isconfined to a narrow bandwidth, very small changes in the opticalcharacteristics of the surface manifest themselves in easily observedchanges in reflected wavelength spectra. The narrow reflection bandwidthprovides a surface adsorption sensitivity advantage compared toreflectance spectrometry on a flat surface.

A SRVD biosensor reflects light predominantly at a first single opticalwavelength when illuminated with a broad band of optical wavelengths,and reflects light at a second single optical wavelength when one ormore specific binding substances are immobilized on the reflectivesurface. The reflection at the second optical wavelength results fromoptical interference. A SRVD biosensor also reflects light at a thirdsingle optical wavelength when the one or more specific bindingsubstances are bound to their respective binding partners, due tooptical interference.

Readout of the reflected color can be performed serially by focusing amicroscope objective onto individual microarray spots and reading thereflected spectrum with the aid of a spectrograph or imagingspectrometer, or in parallel by, for example, projecting the reflectedimage of the microarray onto an imaging spectrometer incorporating ahigh resolution color CCD camera.

A SRVD biosensor can be manufactured by, for example, producing a metalmaster plate, and stamping a relief volume diffractive structure into,for example, a plastic material like vinyl. After stamping, the surfaceis made reflective by blanket deposition of, for example, a thin metalfilm such as gold, silver, or aluminum. Compared to MEMS-basedbiosensors that rely upon photolithography, etching, and wafer bondingprocedures, the manufacture of a SRVD biosensor is very inexpensive.

6. Liquid-Containing Vessels

A SWS or SRVD biosensor embodiment can comprise an inner surface. In onepreferred embodiment, such an inner surface is a bottom surface of aliquid-containing vessel. A liquid-containing vessel can be, forexample, a microtiter plate well, a test tube, a petri dish, or amicrofluidic channel. In one embodiment, a SWS or SRVD biosensor isincorporated into a microtiter plate.

For example, a SWS biosensor or SRVD biosensor can be incorporated intothe bottom surface of a microtiter plate by assembling the walls of thereaction vessels over the resonant reflection surface, so that eachreaction “spot” can be exposed to a distinct test sample. Therefore,each individual microtiter plate well can act as a separate reactionvessel. Separate chemical reactions can, therefore, occur withinadjacent wells without intermixing reaction fluids and chemicallydistinct test solutions can be applied to individual wells.

Several methods for attaching a biosensor of the invention to the bottomsurface of bottomless microtiter plates can be used, including, forexample, adhesive attachment, ultrasonic welding, and laser welding.

The most common assay formats for pharmaceutical screening laboratories,molecular biology research laboratories, and diagnostic assaylaboratories are microtiter plates. The plates are standard-sizedplastic cartridges that can contain 96, 384, or 1536 individual reactionvessels arranged in a grid. Due to the standard mechanical configurationof these plates, liquid dispensing, robotic plate handling, anddetection systems are designed to work with this common format. Abiosensor incorporating an embodiment of the present invention can beincorporated into the bottom surface of a standard microtiter plate.See, e.g., FIGS. 8A & 8B.

For example, FIG. 8A illustrates a microtiter plate 93. The microtiterplate 93 is a bottomless microtiter plate having a plurality of holes 93(a). The plurality of holes, preferably arranged in an array, extendfrom a top surface 96 to a bottom surface 95 of the microtiter plate 93.A microarray slide 94 (FIG. 8B) is provided along the plate bottomsurface 95. The slide acts as a resonant reflection biosensor surfacedue to the incorporation of structure in the bottom surface inaccordance with FIG. 4 or 5, described previously.

Because the biosensor surface can be fabricated in large areas, andbecause a readout system incorporating one aspect of the presentinvention does not make physical contact with the biosensor surface, aplurality of individual biosensor areas can be defined.

7. Holding Fixtures

A number of biosensors that are, for example, about 1 mm² to about 5mm², and preferably less than about 3×3 mm² can be arranged onto aholding fixture that can simultaneously dip the biosensors into separateliquid-containing vessels, such as wells of a microtiter plate, forexample, a 96-, 384-, or 1536-well microtiter plate. Other types ofliquid containing vessels could also be) used including a micro fluidicdevice, a microarray chip, a petri dish, a microscope slide, and aflask.

FIG. 9 illustrates an embodiment of a holding fixture 100. Fixture 100includes a plurality of wells 104. In this embodiment, the fixtureincludes 96 wells arranged in an array comprising 8 rows and 12 columns.Each well 104 includes a plurality of microtiter plate spots 102. Where,for example, the biosensor includes 96 wells, a total of 4800 platespots are provided. (4800=96×50). Therefore, with the embodimentillustrated in FIG. 9, this holding fixture 100 could be dipped into a96-well plate and perform 4800 assays.

Each of the biosensors can contain multiple distinct locations. Aholding fixture has one or more attached biosensors so that eachindividual biosensor can be lowered into a separate, liquid-containingvessel. A holding fixture can comprise plastic, epoxy or metal. Forexample, 50, 96, 384, or 1,000, or 1,536 biosensors can be arranged on aholding fixture, where each biosensor has 25, 100, 500, or 1,000distinct locations. As an example, where 96 biosensors are attached to aholding fixture and each biosensor comprises 100 distinct locations,9600 biochemical assays can be simultaneously performed.

8. Methods of using SWS and SRVD Biosensors

The disclosed SWS and SRVD biosensors can be used to study one or anumber of specific binding substance/binding partner interactions inparallel. Binding of one or more specific binding substances to theirrespective binding partners can be detected, without the use of labels.Detection occurs by applying one or more binding partners to a SWS orSRVD biosensor that have one or more specific binding substancesimmobilized on their surfaces. A SWS biosensor is illuminated with lightand a maxima in reflected wavelength, alternatively or a minima intransmitted wavelength is detected from the biosensor. If one or morespecific binding substances have bound to their respective bindingpartners, then the reflected wavelength of light is shifted as comparedto a situation where one or more specific binding substances have notbound to their respective binding partners. The shift is detected by aspectrographic device as described in further detail. Where a SWSbiosensor is coated with an array of distinct locations containing theone or more specific binding substances, then a maxima in reflectedwavelength or minima in transmitted wavelength of light is detected fromeach distinct location of the biosensor.

A SRVD biosensor is illuminated with light after binding partners havebeen added and the reflected wavelength of light is detected from thebiosensor. Where one or more specific binding substances have bound totheir respective binding partners, the reflected wavelength of light isshifted.

In an alternative embodiment, a variety of specific binding substances,for example, antibodies, can be immobilized in an array format onto abiosensor of the invention. The biosensor is contacted with a testsample of interest comprising binding partners, such as proteins. Onlythe proteins that specifically bind to the antibodies immobilized on thebiosensor remain bound to the biosensor. Such an approach is essentiallya large-scale version of an enzyme-linked immunosorbent assay. However,in this embodiment, the use of an enzyme or fluorescent label is notrequired.

The activity of an enzyme can be detected by applying one or moreenzymes to a SWS or SRVD biosensor to which one or more specific bindingsubstances have been immobilized. The biosensor is washed andilluminated with light. The reflected wavelength of light is detectedfrom the biosensor. Where the one or more enzymes have altered the oneor more specific binding substances of the biosensor by enzymaticactivity, the reflected wavelength of light is shifted.

Additionally, a test sample, for example, cell lysates containingbinding partners can be applied to a biosensor of the invention,followed by washing to remove unbound material. The binding partnersthat bind to a biosensor can be eluted from the biosensor and identifiedby, for example, mass spectrometry. Optionally, a phage DNA displaylibrary can be applied to a biosensor of the invention followed bywashing to remove unbound material. Individual phage particles bound tothe biosensor can be isolated and the inserts in these phage particlescan then be sequenced to determine the identity of the binding partner.

For the above applications, and in particular proteomics applications,the ability to selectively bind material, such as binding partners froma test sample onto a preferred biosensor, followed by the ability toselectively remove bound material from a distinct location of thebiosensor for further analysis is advantageous. Biosensors may also becapable of detecting and quantifying the amount of a binding partnerfrom a sample that is bound to a biosensor array distinct location bymeasuring the shift in reflected wavelength of light. For example, thewavelength shift at one distinct biosensor location can be compared topositive and negative controls at other distinct biosensor locations todetermine the amount of a binding partner that is bound to a biosensorarray distinct location.

9. SWS and Electrically Conducting Material

An alternative biosensor embodiment structure is provided that enables abiosensor array to selectively attract or repel binding partners fromindividual distinct locations on a biosensor. As is well known in theart, an electromotive force can be applied to biological molecules suchas nucleic acids and amino acids subjecting them to an electric field.Because these molecules are electronegative, they are attracted to apositively charged electrode and repelled by a negatively chargedelectrode.

In one embodiment of the present invention, a grating structure of aresonant optical biosensor is provided with an electrically conductingmaterial rather than an electrically insulating material. An electricfield is applied near the biosensor surface. Where a grating operates asboth a resonant reflector biosensor and as an electrode, the gratingcomprises a material that is both optically transparent near theresonant wavelength, and also has low resistivity. In one biosensorembodiment, the material is indium tin oxide, InSn_(x)O_(1-x) (ITO). ITOis commonly used to produce transparent electrodes for flat paneloptical displays, and is therefore readily available at low cost onlarge glass sheets. The refractive index of ITO can be adjusted bycontrolling x, the fraction of Sn that is present in the material.Because the liquid test sample solution has mobile ions (and willtherefore be an electrical conductor), the ITO electrodes are coatedwith an insulating material. In a preferred resonant optical biosensorembodiment, a grating layer is coated with a layer with lower refractiveindex material. Materials such as cured photoresist (n=1.65), curedoptical epoxy (n=1.5), and glass (n=1.4-1.5) are strong electricalinsulators that also have a refractive index that is lower than ITO(n=2.0-2.65). A cross-sectional diagram of a biosensor that incorporatesan ITO grating is shown in FIG. 16.

As illustrated in FIG. 16, n₁ represents the refractive index of anelectrical insulator 222. n₂ represents the refractive index of atwo-dimensional grating 224. t₁ represents the thickness of theelectrical insulator 222. t₂ represents the thickness of thetwo-dimensional grating 224. n_(bio) represents the refractive index ofone or more specific binding substances 228 and t_(bio) represents thethickness of the one or more specific binding substances 228.

A grating can be a continuous sheet of ITO contains an array ofregularly spaced holes. The holes are filled in with an electricallyinsulating material, such as cured photoresist. The electricallyinsulating layer overcoats the ITO grating so that the upper surface ofthe structure is completely covered with electrical insulator, and sothat the upper surface is substantially flat. When the biosensor 220 isilluminated with light 230, a resonant grating effect is produced on thereflected radiation spectrum 225. The depth and the period of thegrating 224 are less than the wavelength of the resonant grating effect.

A single electrode can comprise a region containing a plurality ofgrating periods. For example, FIG. 10 illustrates an embodiment of anarray of biosensor electrodes 110. In this preferred embodiment, thearray of biosensor electrodes 110 includes 9 grating periods 112 (a-i).Providing a plurality of separate grating regions 112 (a-i) on a singlesubstrate surface creates an array of biosensor electrodes. Electricalcontact to regions 112(a-i) is provided using electrically conductingtraces 114 (a-i). Traces 114 (a-i) are preferably constructed from thesame material as a conductor within the biosensor electrode 110.Conducting traces 114 (a-i) are coupled to a voltage source 115. Voltagesource 115 applies an electrical potential to the various gratingregions.

FIG. 11 illustrates a SEM photograph 116 illustrating a plurality ofseparate grating regions 117 (a-f) of an array of biosensor electrodes,such as the electrode illustrated in FIG. 10.

To apply an electrical potential to a biosensor that is capable ofattracting or repelling a molecule near the electrode surface, abiosensor upper surface can be immersed in a liquid sample.

FIG. 12(a) illustrates an embodiment of a biosensor upper surfaceimmersed in a liquid sample 130. The biosensor includes a substrate 124,an ITO rating 125, and an insulator 126. A “common” electrode 122 can beplaced within the sample liquid 130. A voltage from source 123 can beapplied between one selected biosensor electrode region 125 and thecommon electrode. In this manner, one, several, or all electrodes may beactivated or inactivated simultaneously. FIG. 12(b) illustrates theattraction of electronegative molecules 144 to the biosensor surfacewhen a positive voltage is applied to the electrode. FIG. 12(c)illustrates the application of a repelling force such as a reversedelectrical charge to electronegative molecules 144 using a negativeelectrode voltage 123(b).

10. Detection Apparatus and System

FIG. 13 illustrates one embodiment of a detection system 150. Thedetection system 150 comprises a biosensor 152 having the structure aspreviously herein described, e.g., as in FIG. 4. The detection system150 further includes a light source 154 and spectrometer 160. The lightsource 154 directs light to the biosensor 152. A detector, such as thespectrometer 160, detects light reflected via a collecting fiber 153from the biosensor.

The light source 154 illuminates the biosensor 151 via an illuminatingfiber 152 from a sensor top surface, i.e., the surface to which one ormore specific binding substances are immobilized. Alternatively, thebiosensor 152 may be illuminated from its bottom surface. By measuringthe shift in resonant wavelength at each distinct location of thebiosensor 152, it is possible to determine which distinct locations havebinding partners bound to them. The extent of the shift can be used todetermine the amount of binding partners in a test sample and thechemical affinity between one or more specific binding substances andthe binding partners of the test sample.

In one embodiment, the biosensor 152 is illuminated twice. A firstreflected measurement determines the reflectance spectra of one or moredistinct locations of a biosensor array with one or more specificbinding substances immobilized on the biosensor. A second measurementdetermines the reflectance spectra after one or more binding partnersare applied to a biosensor. The difference in peak wavelength betweenthese two measurements is a measurement of the amount of bindingpartners that have specifically bound to a biosensor or one or moredistinct locations of a biosensor. This method of illumination canaccount for small nonuniformities in a surface of a biosensor, resultingin regions with slight variations in the peak resonant wavelength. Thismethod can detect varying concentrations or molecular weights ofspecific binding substances immobilized on a biosensor.

Computer simulation can be used to determine the expected dependencebetween a peak resonance wavelength and an angle of incidentillumination. For example, referring to FIG. 1, substrate 12 may bechosen as glass (n_(substrate)=1.50). The grating 16 could be atwo-dimensional pattern of silicon nitride squares (t₂=180 nm, n₂=2.01(n=refractive index), k₂=0.001 (k=absorption coefficient)) with a periodof 510 nm, and a filling factor of 56.2% (i.e., 56.2% of the surface iscovered with silicon nitride squares while the rest is the area betweenthe squares). The areas between silicon nitride squares may be filledwith a lower refractive index material. The same material covers thesquares and provides a uniformly flat upper surface. In this embodiment,a glass layer may be selected (n₁=1.40) such that it covers the siliconnitride squares by t₂=100 nm.

The reflected intensity as a function of wavelength was modeled usingGSOLVER software. Such software utilizes full 3-dimensional vector codeusing hybrid Rigorous Coupled Wave Analysis and Modal analysis. GSOLVERcalculates diffracted fields and diffraction efficiencies from planewave illumination of arbitrarily complex grating structures. Theillumination can be from any incidence and any polarization.

FIG. 14 illustrates a graphical representation 164 of a resonancewavelength of a biosensor measured as a function of incident angle of anillumination beam. The simulation demonstrates that there is acorrelation between the angle of incident light, and the measured peakwavelength. This result implies that the collimation of the illuminatingbeam, and the alignment between the illuminating beam and the reflectedbeam affects the measured resonant peak linewidth. If the collimation ofthe illuminating beam is poor, a range of illuminating angles will beincident on the biosensor surface, and a wider resonant peak will bemeasured than if purely collimated light were incident.

Because the lower sensitivity limit of a biosensor is related to theability to determine a peak maxima, it is important to measure a narrowresonant peak. Therefore, the use of a collimating illumination systemprovides increased sensitivity.

It may be desirable for the illuminating and collecting fiber probes tospatially share the same optical path. Several methods may be used toco-locate illuminating and collecting optical paths. For example, onemethod includes a single illuminating fiber, which may be coupled at itsfirst end to a light source that directs light at the biosensor, and asingle collecting fiber, which may be coupled at its first end to adetector that detects light reflected from the biosensor. Each can becoupled at their second ends to a third fiber probe such that this thirdfiber acts as both an illuminator and a collector. The third fiber probeis oriented at a normal angle of incidence to the biosensor and supportscounter-propagating illuminating and reflecting optical signals.

FIG. 15 illustrates an alternative embodiment of a detection system 170.In this alternative embodiment, the detection system 170 includes a beamsplitter 174. The beam splitter 174 enables a single illuminating fiber178, which is optically coupled to a light source, to be oriented at a90 degree angle to a collecting fiber 176. The collecting fiber 176 isoptically coupled to a detector. Light is directed through theilluminating fiber probe into the beam splitter, which directs lighttoward the biosensor 172. The reflected light is directed back into thebeam splitter 174, which then directs reflected light into thecollecting fiber probe 176.

A beam splitter allows the illuminating light and the reflected light toshare a common optical path between the beam splitter and the biosensor.

FIG. 17 illustrates an optical fiber probe measuring apparatus 202. FIG.17 is a basic design for a PWV detector that can be adapted to a varietyof possible instrumentation configurations. Generally, the measuringapparatus 202 includes an instrument that detects biochemicalinteractions occurring on a surface 215 of an optical biosensor 211. Aspreviously detailed above, the biosensor 211 may be embedded within abottom portion of a conventional microtiter plate. In one embodiment,the measuring apparatus 202 illuminates the biosensor surface 215 bycasting a spot of collimated white light onto the sensor structure 211.

The measuring apparatus 202 collects light reflected from theilluminated biosensor surface. Preferably, the apparatus illuminates andgathers light from multiple locations on the biosensor surfacesimultaneously. In another embodiment, the apparatus 202 scans thedetection head 209 of a dual illumination probe across the biosensorsurface. Based on the reflected light, the apparatus measures certainvalues, such as the peak wavelength values (PWV's), of a plurality oflocations within the biosensor embedded microtiter plate.

The biosensor can be incubated in a incubation enclosure and moved to aposition for reading. One possible configuration of an instrument thatperforms incubation and reading is set forth below. Incubation may occurat a user determined temperature. An instrument incorporating themeasuring apparatus may also provide a mechanism for mixing sampleswithin a microtiter plate well while the optical sensor resides insidethe apparatus. The mixing could take the form of a shaking mechanism orother type of system.

As illustrated in FIG. 17, measuring apparatus 202 includes a whitelight source 205, an optical fiber probe 208, a collimating lens 210,and a spectrometer 212. A liquid test sample is placed in the spacebetween the structures 216 and 217 for binding to receptors on thesurface 215. The measuring apparatus 202 measures biochemicalinteractions occurring on a surface 215 of the optical device 211.Optical device 211 may have the characteristics of the biosensor devicesas herein previously described, such as a well of microtiter platemodified in accordance with FIGS. 8A and 8B. Advantageously, thesemeasurements occur without the use of fluorescent tags or colorimetriclabels.

As described in more detail above, the surface 215 of the optical device211 contains an optical structure such as shown in FIG. 17. Whenilluminated with collimated white light generated by light source 205,optical device surface 215 is designed to reflect only a narrow band ofwavelengths. This narrow band of wavelengths is described as awavelength peak. The “peak wavelength value” (i.e., PWV) changes whenbiological material is deposited or removed from the sensor surface 215.That is, the PWV changes when a biological material is deposited betweenthe structures 216 and 217.

The measuring apparatus 202 illuminates distinct locations on theoptical device surface with collimated white light, and then collectsthe reflected light. The collected light is gathered into a wavelengthspectrometer 212 for processing, including generation of a PWV.

The measuring apparatus 202 utilizes at least one optical fiber probe208. Referring now to both FIGS. 17 and 18(a), optical fiber probe 208can comprise both an illuminating fiber 218 and a detecting fiber 220.The illuminating fiber 218 is optically coupled to the white lightsource 205 and terminates at a probe head 209 of the optical fiber probe208. Detecting fiber 220 is optically coupled to a spectrometer 212 andterminates at the probe head 209 of the optical fiber probe 208. In thisembodiment, the spectrometer 212 is a single-point spectrometer.

The light source generates white light. The illuminating fiber 218directs an area of collimated light, via the collimating lens 210, on asensor surface, preferably a bottom sensor surface. Preferably, theilluminating fiber is bundled along with the second detecting fiber andcontained in a unitary optical fiber probe, such as fiber optic probe208. This detecting fiber 220 is utilized to collect light reflectedfrom the biosensor 211. The detecting fiber 220 channels the reflectedlight to the spectrometer unit 212, preferably a wavelengthspectrometer. The spectrometer unit 212 processes the reflected light todetermine a resonance peak wavelength value (i.e., PWV) of the reflectedlight.

In one preferred embodiment of a measuring apparatus, white light source205 illuminates a ˜1 millimeter (mm) diameter region of the gratingregion 215 through a 400 micrometer diameter fiber optic and thecollimating lens 210 at nominally normal incidence through the bottom ofa microtiter plate. Such a microtiter plate could have a standard 96-,384-, or 1526-well microtiter plate format, but with a biosensorattached to the bottom.

The measuring apparatus 202 illustrated in FIG. 17 includes asingle-point optical spectrometer included in the spectrometer unit 212.Preferably, the spectrometer unit 212 includes a commercially availableoptical spectrometer such as those spectrometers commercially availablefrom Ocean Optics, Inc. Spectrometer 212 includes a diffraction gratingand a linear or one dimensional charge coupled device (CCD). Thediffraction grating breaks the incident radiation into its componentspectra. The boundaries of the spectra may generally range from 680nanometers (um) to. 930 nm for the device.

The incident radiation impinges a linear (or one dimensional) CCD. Inone embodiment, this one dimensional CCD has 2048 pixels. The CCDconverts incident radiation into an electric charge. There is a generalrelationship between the incident radiation and the resulting electriccharge: the greater the number of incident photons, the greater theamount of charge the pixels in the CCD accumulate.

Each pixel in the CCD images a separate wavelength of light due to thespatial separation provided by the grating in the spectrometer and thedistance between the grating and the CCD. The gap in wavelength imagedby each pixel may range from about 0.13 to about 0.15 nm. Therefore, thefirst pixel in the CCD images light at a wavelength of 680 nm, thesecond pixel at 680.13 nm, the third pixel at 680.27 nm, etc. The 2048thpixel, therefore, images light at approximately 930 nm.

The CCD readout consists of an analog voltage signal for each pixel.These analog voltage signals are converted to a digital signal, rangingin value from 0 to 4,095. A digital value of 0 implies that there is nosignal (i.e., there is no incident radiation). Conversely, a digitalvalue of 4,095 implies that the pixel is saturated with incidentradiation.

A general arrangement of the spectrometer 212 of FIG. 7 is provided inFIG. 18(b). For ease of explanation, the imaging optics inside thespectrometer are not shown in FIG. 18(b). As illustrated in FIG. 18(b),light 221 is incident on the spectrographic grating 222. This light isthen reflected in a dispersed fashion 223, 225 along a surface 224 ofthe CCD 226 with the separation of light in accordance with the shorterwavelength λ₁ imaged at a first end of the CCD and the longer wavelengthλ₂ imaged at a second end of the CCD.

FIG. 18(c) illustrates a readout of the CCD (Y axis) as a function ofpixel number. The curve 229 indicates that there is a peak 231 in pixeloutput for a particular wavelength λ peak. λ peak is the peak wavelengthvalue (PWV) referred to herein elsewhere.

Through calibration, one can determine the relationship betweenwavelength λ and the CCD pixel number, the pixel number beingrepresented in FIG. 18(c) along the x-axis. Commercially availablewavelength spectrometers, such as the spectrometers commerciallyavailable from Ocean Optics, Inc., are provided with informationdetailing a spectrometer's calibration.

For a given microtiter plate well illuminated by the measuring apparatus202, the spectrometer provides a curve of CCD readout as a function of λor pixel number. Thus, an instrument will generate data similar to thatshown in FIG. 18 c and determine PWV for each well or detection locationon the sensor.

In an alternative embodiment, a measuring apparatus is provided thatsimultaneously illuminates and measures a plurality of optical devicesurface regions. In such an alternative embodiment, a plurality of dualprobe fibers are utilized. For example, FIG. 19 illustrates analternative embodiment of a measuring apparatus 260.

Measuring apparatus 260 includes a plurality of dual fiber probes262(a-h) for illuminating and detecting light reflected from opticaldevice surface. In this embodiment, the apparatus 260 includes eight (8)dual fiber probes designated generally as 262(a-h). However, as those ofordinary skill will realize, measuring apparatus 260 could includealternative dual fiber probe arrangements.

Preferably, dual probe fibers 262(a-h) function as both illuminators anddetectors for a single row of microtiter wells provided in bottomlessmicrotiter plate 267. As illustrated, liquid 268 may be provided in afirst microtiter plate well 270. For a conventional 96-well microtiterplate, there would be 8 microtiter wells 268(a-h) in this row.

In one embodiment, the measuring apparatus 260 includes a singlespectrometer included in spectrometer unit 263 and a single light source261. Alternatively, measuring apparatus 260 could include a separatespectrometer and a separate CCD for each dual probe fiber: i.e., thereare 8 spectrometers and 8 CCDs included in the spectrometer unit 263.Alternatively, the measuring apparatus 260 could include a separatelight source for each dual probe in light source unit 261. Theprocessing, measurement, and readout of reflected light for each well issimilar to the processing and readout provided in FIG. 18(c).

The measurement system 260 illustrated in FIG. 19 includes a pluralityof dual fiber probes 262. Each dual fiber probe operates in a similarfashion as the dual probe illustrated in FIG. 17 and as described above.

Preferably, an algorithm processing the curve illustrated in FIG. 18(c)determines a peak wavelength value with sub-pixel resolution. Sub-pixelresolution is generally preferred since this type of resolution providespeak wavelength values with a precision greater than the CCD pixelseparation (i.e., 0.13 to 0.15 nm). A presently preferred algorithm isset forth in section 12 below.

In the dual fiber probe embodiment illustrated in FIG. 19, each dualprobe 262 (a-h) includes an illuminating fiber and a detecting fiber. Inthis preferred embodiment, the apparatus 260 is configured to includeeight dual fiber probes and therefore provides a separate readout perdual probe. In such a configuration, eight dual fiber probessimultaneously illuminate a single row of a standard microtiter plateand measure light reflected from this illuminated row. As those ofordinary skill will realize, other multiple probe configurations mayalso be utilized.

The plurality of dual probes may be arranged side by side in a linearfashion. By utilizing such a linear arrangement, a plurality of dualprobes can simultaneously illuminate and then read out a plurality ofsensor surface locations. For example, a linear probe arrangement may beutilized to illuminate and then read an entire row or an entire columnof a microtiter plate. In this preferred embodiment, each dual probehead contains two optical fibers. The first fiber is connected to awhite light source to cast a small spot of collimated light on thesensor surface. The second fiber reflects the reflected radiation andsupplies it to a spectrometer. After one row is illuminated, relativemotion occurs between the detector probes and the sensor (microtiterplate) and the next row or column of the sensor is read. The processcontinues until all rows (or columns) have been read.

As will be described in further detail below, in one embodiment of themeasuring apparatus, a microtiter plate is placed on a linear motionstage. The linear motion stage moves the microplate in a specified,linear scan direction. As the microtiter plate is moved in this scandirection, each microplate column is sequentially illuminated. Theresulting reflected light is measured. In one preferred embodiment, ascan of a conventional 96-well microtiter plate may take approximately15 to 30 seconds to illuminate and measure the resultant reflectedspectrum.

In yet another alternative embodiment, an imaging apparatus utilizes aspectrometer unit that comprises an imaging spectrometer. For example,FIG. 20 illustrates an alternative embodiment of a biosensor instrumentsystem 319. The instrument system 319 includes an imaging spectrometer332. One advantage of the imaging spectrometer system is that suchimaging systems reduce the amount of time for generating a PWV image.Another advantage is to study biological binding of an area in anon-uniform fashion.

The instrument system 319 illustrated in FIG. 20 includes a collimatedwhite light source 320, a beam splitter 324, and an imaging spectrometer330. Beam splitter 324 redirects the collimated light 322 towards abiosensor in accordance with principles discussed previously. In oneembodiment, the biosensor 319 is a conventional microarray chip 328.More preferably, the microarray chip could comprise a plurality of wellsor spots arranged in an array of uniform rows and columns.

Imaging spectrometer 332 is used to generate a PWV image for each of thereceptor locations (spots) contained in a microarray chip 328. In thisembodiment, the microarray chip 328 comprises a plurality of spotwarranged in eight (8) rows, where each row contains six (6) spotw. Forexample, a first microarray chip row 331 contains six spotw 331(a-f).Similarly, a second microarray chip row 333 contains six spotw 333(a-f).All the spotw of each row can be simultaneously illuminated incidentlight 332. As those of ordinary skill in the art will recognize, thetotal number of spots on a microarray chip can be much larger, routinelyin the tens of thousands.

Light 322 is collimated and directed towards the beam splitter 324. Thebeam splitter 324 allows the collimated light 322 and the reflectedlight 334 to share a common optical path between the beam splitter andthe microarray chip 328.

Preferably, the collimated light is directed at the sensor bottomsurface 342 at normal incidence via the beam splitter 324. Normallyreflected light 334 is collected into an input 335 of the imagingspectrometer 332.

At the microarray chip 328, redirected light 330 illuminates an imagingarea 332 along a bottom surface 342 of the microarray chip 328. Theredirected light 330 is used to illuminate this imaging area 332. Inthis embodiment, the illuminated imaging area 332 may be defined by aplurality of spotw 331 along the microarray chip 328. Preferably, theilluminated imaging area 332 is defined by either a complete row or acomplete column of spotw contained on the microarray chip 328. Forexample, illuminated imaging area 332 could be defined to include thefirst microarray chip row 331 or row 333.

The bottom surface 342 of the microarray chip 328 is scanned in asequential manner. To accomplish a complete scan of the bottom surface342, the microarray chip 328 is transported along a scan direction “A.”As the microarray chip 328 is transported along this scan direction, thecollimated light 330 traverses along the complete length of the bottomsurface 342 of the microarray chip. In this manner, the instrumentsystem 319 sequentially illuminates and reads out all of the spotw in amicroarray chip 328. For example, as the chip 328 is moved in the “A”scan direction, chip row 333 is first illuminated and read out, and thenchip row 331 is illuminated and then read out. The process continuesuntil all rows are read.

Preferably, the illuminated imaging area includes all the spotw along amicroarray row. For example, at a given point in time, the redirectedlight may illuminate a first row of spotw on the 8×6 microarray chip.Consequently, light 330 illuminates all six spotw 331(a-f) contained inthe first row 331 of the microarray chip 328. As the microarray chip 328is transported along scan direction A, the collimated white light source320 simultaneously illuminates all of the spotw contained in the nextmicroarray row. To complete the read out of the entire microarray chip,each of the eight (8) rows of the microarray chip are sequentiallyilluminated.

The spectrometer unit 332 preferably comprises an imaging spectrometercontaining a two-dimensional Charge Coupled Device (CCD) camera and adiffraction grating. The reflected light 334 containing the biosensorresonance signal for each spot is diffracted by the grating in thespectrometer unit. The diffraction produces a spatially segregatedwavelength spectra for each point within the illuminated area. (See,e.g., FIG. 18(c). The wavelength spectrum has a second spatial componentcorresponding to the direction transverse to the scan direction “A.”This second spatial component is subdivided into discrete portionscorresponding to spotw in this transverse direction.

For example, if the imaging spectrometer includes a CCD camera thatcontains 512×2048 imaging elements, then an illuminating line isspatially segregated into 512 imaging elements or points. A wavelengthspectra is measured for each of the 512 imaging elements or points alongthe orthogonal axis of the CCD camera. Where the CCD camera contains512×2048 imaging elements, the CCD would have a resolution of 2048wavelength data points. Using this method, the PWV's of 512 points aredetermined for a single “line” or imaging area across the sensor bottomsurface 342. For a conventional CCD imaging camera typically havingspatial resolution of approximately 10 microns, a 1:1 imaging system iscapable of resolving PWV values on sensor surface 342 with a 10 micronresolution. In order to measure a PWV image of the entire sensor bottomsurface 342, the sensor 328 is transported along an imaging plane (scandirection A), and subsequent line scans are used to construct a PWVimage.

FIGS. 21-24 illustrate various aspects of yet another alternativeembodiment of a measuring instrument 400 which incorporates variousprinciples of FIG. 19. FIG. 21 illustrates a perspective view of themeasuring instrument 400.

Measuring instrument 400 includes a measuring instrument cover 452 and adoor 454. A microplate well plate (or microtiter plate) 456 configuredas a biosensor in accordance with this invention is shown in anextracted position, outside an incubator assembly 460 incorporated inthe measuring instrument 400. The microplate well plate 456 is held by amicrowell tray 458. The tray 458 may extend out of the incubatorassembly 460 through a door way 453 located at the front of theincubator assembly 460. The incubator assembly 460 allows the tray 458to be maintained at a user defined temperature during microwell trayread out and/or measurement.

In one preferred embodiment, incubator assembly 460 is used forperforming assays at controlled temperatures, typically such controlledtemperatures may range from 4 and 45 degrees Celsius. As will beexplained with reference to FIGS. 22, 23, and 24, a collimator assembly708 is positioned preferably beneath a bottom portion 602 of theincubator assembly 460. During microtiter well illumination andwavelength measurement, the collimator assembly 708 illuminates a bottomsurface 459 of the tray 458.

While the tray 458 remains in an extracted position outside of theincubator assembly 460, the microtiter plate 456 may be placed on orremoved from the tray 458. The plate 456 may be held in the tray 458 viaa set of registration points, spring clips, or other known types ofsecuring means. In FIG. 22, clips 457 are used to hold the plate 456 inthe tray 458.

After the microtiter plate 456 has been loaded with a fluid sample withbiological material to be detected and measured, the tray 458 istransported into the incubator assembly 460. Processing, mixing,heating, and/or readout of the biosensors may then begin, preferablyunder the control of the controller assembly 588 (see FIG. 22).

Once the tray 458 retracts into the incubator assembly 460, the trayremains stationary during illumination and read out. For a readout ofthe microtiter plate 456 to occur, the collimator assembly 708 generatesan illumination pattern that is incident along the bottom surface 459 ofthe plate 456. Preferably, the measuring apparatus 400 generates a beamof light that is incident along an entire row of wells of the plate 456.

Alternatively, the measuring apparatus 400 generates a plurality ofillumination beams that are simultaneously incident on a plurality ofplate wells. The illumination pattern, comprising multiple beams, isgenerated by dual illumination fiber optic probes contained within thecollimator assembly 708. The construction of the probes is as shown isFIG. 17. As previously herein described, the light reflected off of thebiosensor surface may then be detected by the same plurality of probescontained within a collimator assembly 708. This reflected light is thenanalyzed via the spectrometer system 590.

The incubator assembly 460 is provided with a plurality of apertures 764along a bottom incubator assembly structure. As can be seen in FIG. 24,incubator assembly apertures 764 are configured to generally line-up andmatch the well locations 657 on the plate 456 when the plate 456 is in areadout position within the incubator assembly 460. For example, ifthere are 96 wells on the microwell well plate 456, the incubatorassembly bottom portion 602 will be provided with 96 apertures 764.These apertures will be configured in the same type of array as thewells of the well plate (e.g., 8 rows by 6 columns). These apertures 764provide clearance for light generated by collimator assembly 708 toreach the wells from the illuminating probes 709.

To enable user access to the tray and to the plate, the plate tray 458extends out of the measuring apparatus 400. The tray 458 can beretracted into the apparatus 400 and the door cover 454 closed to beginmicroplate processing. Such processing could include mixing liquid inthe microtiter wells, heating deposited liquids to a predeterminedtemperature, illumination of the microplate 456, and processing variousreflected illumination patterns.

FIG. 22 illustrates a perspective view 580 of various internalcomponents of the measuring instrument 400 illustrated in FIG. 21. Asshown in FIG. 22, internal components of the measuring instrument 580include a transition stage assembly 560, heater controller unit 582, acontroller board assembly 588, and a spectrometer unit 590. Thetransition stage assembly 560 includes the incubator assembly 460 andthe collimator assembly 708. The heater controller unit 582, thecontroller board assembly 588, the transition stage assembly 560, andthe spectrometer unit 590 are mounted on a base plate 592. Themicroplate well tray 556 is shown in the retracted position, outside ofthe incubator assembly 460.

The heater controller unit 582 provides temperature control to theincubator assembly 460. The controller board assembly 588 providesfunctional controls for the measuring apparatus including the mixing andother motion controls related to translation stage 560 and tray handling458.

The spectrometer unit 590 contains an appropriate spectrometer forgenerating the PWV data. The design of the spectrometer will varydepending on the illumination source. If the probes of FIG. 17 are used,the spectrometer will ordinarily have the design shown in FIG. 18B.

FIG. 23 illustrates a perspective view of the transition stage assembly560 of the measuring instrument 400 illustrated in FIGS. 21 and 22. FIG.26 illustrates the transition stage assembly 560 of FIG. 23 with anincubator assembly top portion 461 removed (See FIGS. 22 and 23). As canbe seen from FIGS. 23 and 24, the transition stage assembly 560 includesthe microwell tray 458 positioned in the retracted position. Themicrowell tray 458 has a plurality of wells 657, enters the incubationassembly 460 (FIG. 23) to initiate the read out process.

The microwell plate tray 458 is mounted on a top surface 605 of a bottomportion 602 of the incubator assembly 460. Preferably, where themicrotiter tray 456 is a conventional microtiter tray having 96 wells,the bottom portion 602 of the incubator assembly 460 includes 96 holes.The microwell plate tray 458 is positioned over the bottom portion ofthe incubator assembly 602 such that the incubator assembly apparatusessentially matches up with the apertures (wells) contained in themicrowell tray 458. Alternately, bottom portion 602 may contain atransparent section that matches the bottom portion of the plate, or mayeliminate the bottom portion.

During specimen illumination and measurement, the microwell tray 458 ispreferably held in a stationary manner within the incubator assembly 460by the bottom incubator assembly portion 602. During illumination andmeasurement, the collimator assembly 708 is held in a stationary mannerwhile a stepping motor 606 drives the incubator assembly, including theplate, in a linear direction “A”. As the incubator assembly 460 isdriven along direction “A,” the collimator assembly 708 illuminates thebottom surface 459 of microtiter plate 456. The resulting reflectedillumination patterns are detected by the collimator assembly 708. Ahome position sensor 710 is provided as a portion of the translationstage assembly and to determine the position during the illuminationprocess.

The transition stage assembly 760 is provided with a plurality ofelastomer isolators 762. In this embodiment, a total of six elastomerisolators are used to provide isolation and noise reduction duringillumination and read out.

As can be seen from FIGS. 23 and 24, the collimator assembly 708 ispositioned below a bottom surface 603 of the incubator portion bottomportion 602. Preferably, the collimator assembly 708 includes aplurality of dual fiber probe heads 709. In the embodiment illustratedin FIG. 24, the collimator assembly 708 includes 8 dual fiber probeheads 709. These dual fiber probes could have a probe head configurationsimilar to the fiber optic probes illustrated in FIG. 19 and aspreviously described. Alternatively, the collimator assembly 708 couldinclude a PWV imaging system such as the PWV imaging system illustratedin FIG. 20.

For ease of explanation, only the bottom plate 602 of the incubatorassembly 460 is shown is FIG. 24. The incubator assembly bottom portion602 is provided with a plurality of apertures 764. Preferably, where themicrowell plate 456 is provided with an 8×12 array of wells such asillustrated in FIG. 24, the incubator assembly bottom portion 602 willalso include an 8×12 array of 96 apertures. These apertures willessentially match the 96 wells on the microwell plate 456. In thismanner, the collimated white light generated by the collimator assembly708 propagates through a first surface 603 along the incubator assemblybottom portion 602, and exit a second surface or top surface 605 ofincubator assembly bottom portion 602. The collimated light can thenilluminate a bottom well portion of the microwell plate 456.Alternately, bottom portion 602 may contain a transparent section thatmatches the bottom portion of the plate, or may eliminate the bottomportion.

Referring to FIGS. 23 and 24, a drive motor 606 is provided for drivingthe incubator assembly during well scanning. A home position sensor 710is provided as a stop measuring during the translation stage. The platehandling stage uses a stepping motor 702 to drive a rack-and-pinionmechanism. The scanning stage uses a stepping motor 606 to drive aleadscrew 559 along translation stage rails 557, 558.

A mixer assembly may be used for mixing the liquid in the wells. In thepresent invention, a mixing mechanism is located between the incubationchamber of the translation stage. Additionally, a mixing mechanism maybe provided in an alternative location.

FIG. 25 illustrates an example of a microarray image. Specifically, FIG.25 illustrates ten spots 800 of human-IgG spotted on a TaO sensorsurface. Each spot is approximately 400-microns in diameter. FIG. 25illustrates the result of subtracting a pre-spotted image from apost-spotted image. The intensity scale conversion factor is illustratedto be a 0.04 nm per display intensity unit, resulting in a detectedwavelength shift of 0.8 nm.

11. Angular Scanning

The proposed detection systems are based on collimated white lightillumination of a biosensor surface and optical spectroscopy measurementof the resonance peak of the reflected beam. Molecular binding on thesurface of a biosensor is indicated by a shift in the peak wavelengthvalue, while an increase in the wavelength corresponds to an increase inmolecular absorption.

As shown in theoretical modeling and experimental data, the resonancepeak wavelength is strongly dependent on the incident angle of thedetection light beam. Because of the angular dependence of the resonancepeak wavelength, the incident white light needs to be collimated.Angular dispersion of the light beam broadens the resonance peak, andcould reduce biosensor detection sensitivity. In addition, the signalquality from the spectroscopic measurement could depend on the power ofthe light source and the sensitivity of the detector. In order to obtaina desirable signal-to-noise ratio, a lengthy integration time for eachdetection location may be required, and therefore lengthen overall timeto readout a biosensor plate. A tunable laser source can be used fordetection of grating resonance, but is generally cost prohibitive.

In one embodiment, these disadvantages are addressed by using a laserbeam for illumination of a biosensor, and a light detector formeasurement of reflected beam power. A scanning mirror device can beused for varying the incident angle of the laser beam, and an opticalsystem is used for maintaining collimation of the incident laser beam.See, e.g., “Optical Scanning” (Gerald F. Marchall ed., Marcel Dekker(1991). Any type of laser scanning can be used. For example, a scanningdevice that can generate scan lines at a rate of about 2 lines to about1,000 lines per second is useful in the invention. In one embodiment ofthe invention, a scanning device scans from about 50 lines to about 300lines per second.

In one embodiment, the reflected light beam passes through part of thelaser scanning optical system, and is measured by a single lightdetector. The laser source can be a diode laser with a wavelength of,for example, 780 nm, 785 nm, 810 nm, or 830 nm. Laser diodes such asthese are readily available at power levels up to 150 mW, and theirwavelengths correspond to high sensitivity of Si photodiodes. Thedetector thus can be based on photodiode biosensors.

In another detection system embodiment, while a scanning mirror changesits angular position, the incident angle of the laser beam on thesurface changes by nominally twice the mirror angular displacement. Thescanning mirror device can be a linear galvanometer, operating at afrequency of about 2 Hz up to about 120 Hz, and mechanical scan angle ofabout 10 degrees to about 20 degrees. In this example, a single scan canbe completed within about 10 msec. A resonant galvanometer or a polygonscanner can also be used.

The angular resolution depends on the galvanometer specification, andreflected light sampling frequency. Assuming galvanometer resolution of30 arcsec mechanical, corresponding resolution for biosensor angularscan is 60 arcsec, i.e. 0.017 degree. In addition, assume a samplingrate of 100 ksamples/sec, and 20 degrees scan within 10 msec. As aresult, the quantization step is 20 degrees for 1000 samples, i.e. 0.02degree per sample. In this example, a resonance peak width of 0.2degree, as shown by Peng and Morris (Experimental demonstration ofresonant anomalies in diffraction from two-dimensional gratings, OpticsLett., 21:549 (1996)), will be covered by 10 data points, each of whichcorresponds to resolution of the detection system.

The advantages of such a detection system includes: increasedcollimation of incident light by a laser beam, high signal-to-noiseratio due to high beam power of a laser diode, low cost due to a singleelement light detector instead of a spectrometer, and high resolution ofresonance peak due to angular scanning.

12. Mathematical Resonant Peak Determination

The sensitivity of a biosensor is determined by the shift in thelocation of the resonant peak when material is bound to the biosensorsurface. Because of noise inherent in the spectrum, it is preferable touse a procedure for determining an analytical curve—the turning point(i.e., peak) of which is well defined. Furthermore, the peakcorresponding to an analytic expression can be preferably determined togreater than sub-sampling-interval accuracy, providing even greatersensitivity.

One embodiment utilizes a method for determining a location of aresonant peak for a binding partner in a resonant reflectance spectrumwith a colormetric resonant biosensor. The method comprises selecting aset of resonant reflectance data for a plurality of colormetric resonantbiosensors or a plurality of biosensor distinct locations. The set ofresonant reflectance data is collected by illuminating a colormetricresonant diffractive grating surface with a light source and measuringreflected light at a pre-determined incidence. The colormetric resonantdiffractive grating surface is used as a surface binding platform forone or more specific binding substances such that binding partners canbe detected without use of a molecular label.

The step of selecting a set of resonant reflectance data can includeselecting a set of resonant reflectance data:

-   -   a. x_(i) and y_(i) for i=1,2,3, . . . n,    -   b. wherein x_(i) is a first measurement includes a first        reflectance spectra of one or more specific binding substances        attached to the colormetric resonant diffractive grating        surface, y_(i) is a second measurement and includes a second        reflectance spectra of the one or more specific binding        substances after a plurality of binding partners are applied to        colormetric resonant diffractive grating surface including the        one or more specific binding substances, and n is a total number        of measurements collected.

The set of resonant reflectance data includes a plurality of sets of twomeasurements, where a first measurement includes a first reflectancespectra of one or more specific binding substances that are attached tothe colormetric resonant diffractive grating surface and a secondmeasurement includes a second reflectance spectra of the one or morespecific binding substances after one or more binding partners areapplied to the colormetric resonant diffractive grating surfaceincluding the one or more specific binding substances. A difference in apeak wavelength between the first and second measurement is ameasurement of an amount of binding partners that bound to the one ormore specific binding substances. A sensitivity of a colormetricresonant biosensor can be determined by a shift in a location of aresonant peak in the plurality of sets of two measurements in the set ofresonant reflectance data.

A maximum value for a second measurement from the plurality of sets oftwo measurements is determined from the set of resonant reflectance datafor the plurality of binding partners, wherein the maximum valueincludes inherent noise included in the resonant reflectance data. Amaximum value for a second measurement can include determining a maximumvalue y_(k) such that:

-   -   c. (y_(k)>=y_(i)) for all i, ≠k.

The algorithm determines whether the maximum value is greater than apre-determined threshold. This can be calculated by, for example,computing a mean of the set of resonant reflectance data; computing astandard deviation of the set of resonant reflectance data; anddetermining whether ((y_(k)−mean)/standard deviation) is greater than apre-determined threshold. The pre-determined threshold is determined bythe user. The user will determine what amount of sensitivity is desiredand will set the pre-determined threshold accordingly.

If the maximum value is greater than a pre-determined threshold acurve-fit region around the determined maximum value is defined. Thestep of defining a curve-fit region around the determined maximum valuecan include, for example:

-   -   d. defining a curve-fit region of (2w+1) bins, wherein w is a        pre-determined accuracy value;    -   e. extracting (x_(i),k−w<=i<=k+w); and    -   f. extracting (y_(i),k−w<=i<=k+w).

A curve-fitting procedure is performed to fit a curve around thecurve-fit region, wherein the curve-fitting procedure removes apre-determined amount of inherent noise included in the resonantreflectance data. A curve-fitting procedure can include, for example:

-   -   g. computing g_(i)=ln y_(i);    -   h. performing a 2^(nd) order polynomial fit on g_(i) to obtain        g′_(i) defined on    -   i. (x_(i),k−w<=i<=k+w);    -   j. determining from the 2^(nd) order polynomial fit coefficients        a, b and c of for (ax²+bx+c)−; and    -   k. computing y′_(i)=e^(g′i).

The location of a maximum resonant peak is determined on the fittedcurve, which can include, for example, determining a location of maximumreasonant peak (x_(p)(−b)/2a). A value of the maximum resonant peak isdetermined, wherein the value of the maximum resonant peak is used toidentify an amount of biomolecular binding of the one or more specificbinding substances to the one or more binding partners. A value of themaximum resonant peak can include, for example, determining the valuewith of x_(p) at y′_(p).

Alternatively, peak values of the measurement apparatus embodiments maybe derived by the mathematical resonant peak determination described incommonly assigned related copending patent application Ser. No. ______(MBHB 01-1775), the entirety of which is herein incorporated byreference and to which the reader is directed for further information.

One embodiment of the measurement apparatus includes a computer readablemedium having stored therein instructions for causing a processor toexecute a method for determining a location of a resonant peak for abinding partner in a resonant reflectance spectrum with a colormetricresonant biosensor. A computer readable medium can include, for example,magnetic disks, optical disks, organic memory, and any other volatile(e.g., Random Access Memory (“RAM”)) or non-volatile (e.g., Read-OnlyMemory (“ROM”)) mass storage system readable by the processor. Thecomputer readable medium includes cooperating or interconnected computerreadable medium, which exist exclusively on a processing system or to bedistributed among multiple interconnected processing systems that can belocal or remote to the processing system.

The following are provided for exemplification purpose only and are notintended to limit the scope of the invention described in broad termsabove. All references cited in this disclosure are incorporated hereinby reference.

Sensor Readout Instrumentation

In order to detect reflected resonance, a white light source canilluminate an approximately 1 mm diameter region of a biosensor surfacethrough a 400 micrometer diameter fiber optic and a collimating lens.Smaller or larger areas may be sampled through the use of illuminationapertures and different lenses. A group of six detection fibers may bebundled around the illumination fiber for gathering reflected light foranalysis with a spectrometer (Ocean Optics, Dunedin, Fla.). For example,a spectrometer can be centered at a wavelength of 800 nm, with aresolution of approximately 0.14 nm between sampling bins. Thespectrometer integrates reflected signal for 25-75 msec for eachmeasurement. The biosensor sits upon an x-y motion stage so thatdifferent regions of the biosensor surface can be addressed in sequence.

Equivalent measurements can be made by either illuminating the topsurface of device, or by illuminating through the bottom surface of thetransparent substrate. Illumination through the back is preferred whenthe biosensor surface is immersed in liquid, and is most compatible withmeasurement of the biosensor when it is incorporated into the bottomsurface of, for example, a microwell plate.

Mathematical Resonant Peak Determination

This example discusses some of the findings that have been obtained fromlooking at fitting different types of curves to the observed data.

The first analytic curve examined is a second-order polynomial, given byy=ax ² +bx+c

The least-squares solution to this equation is given by the costfunction${\phi = {\sum\limits_{i = 1}^{n}\left( {{ax}_{i}^{2} + {bx}_{i} + c - y_{i}} \right)^{2}}},$

the minimization of which is imposed by the constraints$\frac{\partial\phi}{\partial a} = {\frac{\partial\phi}{\partial b} = {\frac{\partial\phi}{\partial c} = 0.}}$

Solving these constraints for a, b, and c yields $\begin{pmatrix}a \\b \\c\end{pmatrix} = {\begin{pmatrix}{\sum x_{i}^{4}} & {\sum x_{i}^{3}} & {\sum x_{i}^{2}} \\{\sum x_{i}^{3}} & {\sum x_{i}^{2}} & {\sum x_{i}} \\{\sum{x2}} & {\sum x_{i}} & n\end{pmatrix}^{- 1} \cdot {\begin{pmatrix}{\sum{x_{i}^{2}y_{i}}} \\{\sum{x_{i}y_{i}}} \\{\sum y_{i}}\end{pmatrix}.}}$

Empirically, the fitted curve does not appear to have sufficient riseand fall near the peak. An analytic curve that provides bettercharacteristics in this regard is an exponential curve, such as aGaussian curve. A simple method for performing a Gaussian-like fit is toassume that the form of the curve is given byy=e ^(ax) ² ^(+bx+c),in which case the quadratic equations above can be utilized by formingy′, where y′=lny.

Assuming that the exponential curve is the preferred data fittingmethod, the robustness of the curve fit is examined in two ways: withrespect to shifts in the wavelength and with respect to errors in thesignal amplitude.

To examine the sensitivity of the analytical peak location when thewindow from which the curve fitting is performed is altered to fall 10sampling intervals to the left or to the right of the true maxima. Theresulting shift in mathematically-determined peak location is shown inTable 3. The conclusion to be derived is that the peak location isreasonably robust with respect to the particular window chosen: for ashift of approximately 1.5 nm, the corresponding peak location changedby only <0.06 nm, or 4 parts in one hundred sensitivity.

To examine the sensitivity of the peak location with respect to noise inthe data, a signal free of noise must be defined, and then incrementalamounts of noise is added to the signal and the impact of this noise onthe peak location is examined. The ideal signal, for purposes of thisexperiment, is the average of 10 resonant spectra acquisitions.

Gaussian noise of varying degrees is superimposed on the ideal signal.For each such manufactured noisy signal, the peak location is estimatedusing the 2^(nd)-order exponential curve fit. This is repeated 25 times,so that the average, maximum, and minimum peak locations are tabulated.This is repeated for a wide range of noise variances—from a variance of0 to a variance of 750. TABLE 3 Comparison of peak location as afunction of window location Shift Window Peak Location □ = −10 bins771.25-782.79 nm  778.8221 nm □ = 0 bins 772.70-784.23 nm  778.8887 nm □= +10 bins 774.15-785.65 nm 7778.9653 nm

The conclusion of this experiment is that the peak location estimationroutine is extremely robust to noisy signals. In one embodiment, theentire range of peak locations is only 1.5 nm, even with as much randomnoise variance of 750 superimposed—an amount of noise that issubstantially greater that what has been observed on the biosensor thusfar. The average peak location, despite the level of noise, is within0.0.1 nm of the ideal location.

Based on these results, a basic algorithm for mathematically determiningthe peak location of a calorimetric resonant biosensor is as follows:

-   1. Input data x_(i) and y_(i), i=1, . . . ,n-   2. Find maximum    -   a. Find k such that y_(k)≧y_(i) for all i≠k-   3. Check that maximum is sufficiently high    -   a. Compute mean {overscore (y)} and standard deviation σ of        sample    -   b. Continue only if (y_(k)−{overscore (y)})!σ>UserThreshold-   4. Define curve-fit region of 2w+1 bins (w defined by the user)    -   a. Extract x_(i),k−w≦i≦k+w    -   b. Extract y_(i),k−w≦i≦k+w-   5. Curve fit    -   a. g_(i)=ln y_(i)    -   b. Perform 2^(nd)-order polynomial fit to obtain g′_(i) defined        on x_(i),k−w≦i≦k+w    -   c. Polynomial fit returns coefficients a,b,c of form ax²+bx+c    -   d. Exponentiate: y′_(i)=e^(g′) ^(i) ;-   6. Output    -   a. Peak location p given by x_(p)=−b/2a    -   b. Peak value given by y′_(p) (x_(p))

In summary, a robust peak determination routine has been demonstrated;the statistical results indicate significant insensitivity to the noisein the signal, as well as to the windowing procedure that is used. Theseresults lead to the conclusion that, with reasonable noise statistics,that the peak location can be consistently determined in a majority ofcases to within a fraction of a nm, perhaps as low as 0.1 to 0.05 nm.

Those skilled in the art to which the present invention pertains maymake modifications resulting in other embodiments employing principlesof the present invention without departing from its spirit orcharacteristics, particularly upon considering the foregoing teachings.Accordingly, the described embodiments are to be considered in allrespects only as illustrative, and not restrictive, and the scope of thepresent invention is, therefore, indicated by the appended claims ratherthan by the foregoing description. Consequently, while the presentinvention has been described with reference to particular embodiments,modifications of structure, sequence, materials and the like apparent tothose skilled in the art would still fall within the scope of theinvention.

1. An instrument system for detecting a biochemical interaction on abiosensor comprising an array of detection locations, said systemcomprising: a light source for generating collimated white light; a beamsplitter directing said collimated white light towards a surface of asensor corresponding to said detector locations; and a detection systemincluding an imaging spectrometer receiving said reflected light andgenerating an image of said reflected light.
 2. The invention of claim 1wherein said biosensor is a microarray chip.
 3. The invention of claim 2wherein said microarray chip is a conventional microarray chip.
 4. Theinvention of claim 1 wherein said biosensor is transported along a scandirection.
 5. The invention of claim 1 wherein said imaging spectrometergenerates a Peak Wavelength Value image.
 6. The invention of claim 1wherein said directed collimated white light is directed to a pluralityof locations on said surface of said sensor.
 7. The invention of claim 1wherein said directed collimated white light is simultaneously directedto a plurality of locations on said surface of said sensor.
 8. Theinvention of claim 1 wherein said directed collimated white light isdirected to an imaging area on said surface of said sensor.
 9. Theinvention of claim 2 wherein said directed collimated white lightilluminates a plurality of wells of said microarray chip.
 10. Theinvention of claim 1 wherein said imaging spectrometer includes atwo-dimensional Charge Coupled Device camera.
 11. The invention of claim1 wherein said imaging spectrometer includes a diffraction grating. 12.The invention of claim 1 including a software interface, said softwareinterface controlling said imaging spectrometer.
 13. The invention ofclaim 12 wherein said software interface coordinates Peak WavelengthValue determination with an x-y motion stage.
 14. The invention of claim12 wherein said software interface converts measured data into a PeakWavelength Value.
 15. The invention of claim 1, wherein said lightsource illuminates said biosensor from a sensor top surface
 16. Theinvention of claim 1 wherein said light source illuminates saidbiosensor from a sensor bottom surface.
 17. An instrument forcalculating a peak wavelength, said instrument comprising: an incubatorassembly for incubating a biosensor; an optical assembly, said opticalassembly illuminating said biosensor with light and collecting reflectedradiation from said biosensor; a spectrometer receiving said reflectedradiation; and software deriving a peak wavelength from said reflectedand detected wavelength.
 18. The invention of claim 17 wherein saidbiosensor is embedded within a bottom portion of a microtiter plate. 19.The invention of claim 17 wherein said collimator assembly comprises aplurality of fiber optic probes.
 20. The invention of claim 18 whereinsaid plurality of fiber optic probes comprise an illuminating fiberoptic probe for illuminating said biosensor and a detecting fiber opticprobe for detecting said reflected wavelength.
 21. The invention ofclaim 18 wherein said collimator assembly comprises a beam splitter,said beam splitter enables said illuminated light and said reflectedlight to share a common optical path.
 22. The invention of claim 17wherein said collimator assembly includes a collimating lens, saidcollimating lens focuses said white light on said biosensor surface. 23.The invention of claim 17 wherein said biosensor comprises: a firsttwo-dimensional grating comprising a first refractive index material andhaving a top surface and a bottom surface; a substrate layer comprisinga top surface and a bottom surface, wherein said top surface of saidsubstrate supports said bottom surface of said first two-dimensionalgrating; and a second two-dimensional grating comprising a secondrefractive index material and having a top surface and a bottom surface,wherein said bottom surface of said second two-dimensional grating isattached to said bottom surface of said substrate; wherein, when saidbiosensor is illuminated two resonant grating effects are produced insaid reflected radiation spectrum, and wherein said depth and period ofboth of said two-dimensional gratings are less than said wavelength ofsaid resonant grating effects.